Human Secreted Proteins

ABSTRACT

The present invention relates to human secreted polypeptides, and isolated nucleic acid molecules encoding said polypeptides, useful for diagnosing and treating diseases, disorders, and/or conditions (such as immune, cardiovascular, cancer, and other proliferative diseases, disorders, and/or conditions) related to said human secreted proteins. Antibodies that bind these polypeptides are also encompassed by the present invention. Also encompassed by the invention are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating diseases, disorders, and/or conditions related to these novel human secreted proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No. 12/136,548, filed Jun. 10, 2008, which is a divisional of U.S. application Ser. No. 11/781,665, filed Jul. 23, 2007, which is a continuation of U.S. application Ser. No. 10/994,608, filed Nov. 23, 2004, which is a divisional of U.S. application Ser. No. 10/105,299, filed Mar. 26, 2002, which claims the benefit of U.S. Provisional Application No. 60/278,650, filed Mar. 27, 2001; U.S. application Ser. No. 10/105,299 is a continuation-in-part of U.S. application Ser. No. 09/950,082, filed Sep. 12, 2001; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06043, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/167,061, filed Nov. 23, 1999, and 60/124,146, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06012, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/166,989, filed Nov. 23, 1999, and 60/124,093, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06058, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,654, filed Dec. 3, 1999, and 60/124,145, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06044, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,661, filed Dec. 3, 1999, and 60/124,099, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06059, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,622, filed Dec. 3, 1999, and 60/124,096, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06042, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,663, filed Dec. 3, 1999, and 60/124,143, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06014, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,665, filed Dec. 3, 1999, 60/138,598, filed Jun. 11, 1999, and 60/124,095, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06013, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,662, filed Dec. 3, 1999, 60/138,626, filed Jun. 11, 1999, and 60/125,360, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06049, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,667, filed Dec. 3, 1999, 60/138,574, filed Jun. 11, 1999, and 60/124,144, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06057, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,666, filed Dec. 3, 1999, 60/138,597, filed Jun. 11, 1999, and 60/124,142, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06824, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,664, filed Dec. 3, 1999, and 60/125,359, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06765, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,906, filed Dec. 10, 1999, and 60/126,051, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06792, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,980, filed Dec. 10, 1999, and 60/125,362, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06830, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,910, filed Dec. 10, 1999, and 60/125,361, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06782, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,936, filed Dec. 10, 1999, and 60/125,812, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06822, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,916, filed Dec. 10, 1999, and 60/126,054, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06791, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,946, filed Dec. 10, 1999, and 60/125,815, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06828, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,616, filed Dec. 8, 1999, and 60/125,358, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06823, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,623, filed Dec. 8, 1999, and 60/125,364, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/06781, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,617, filed Dec. 8, 1999, and 60/125,363, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07505, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,410, filed Dec. 17, 1999, and 60/126,502, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07440, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,409, filed Dec. 17, 1999, and 60/126,503, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07506, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,412, filed Dec. 17, 1999, and 60/126,505, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07507, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,408, filed Dec. 17, 1999, and 60/126,594, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07535, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,413, filed Dec. 17, 1999, and 60/126,511, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07525, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,549, filed Dec. 22, 1999, and 60/126,595, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07534, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,504, filed Dec. 22, 1999, and 60/126,598, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07483, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,552, filed Dec. 22, 1999, and 60/126,596, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07526, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,550, filed Dec. 22, 1999, and 60/126,600, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07527, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,551, filed Dec. 22, 1999, and 60/126,501, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07661, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,847, filed Jan. 7, 2000, and 60/126,504, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07579, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,853, filed Jan. 7, 2000, and 60/126,509, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07723, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,852, filed Jan. 7, 2000, and 60/126,506, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07724, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,850, filed Jan. 7, 2000, and 60/126,510, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14929, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,851, filed Jan. 7, 2000, and 60/138,573, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07722, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,871, filed Jan. 7, 2000, and 60/126,508, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07578, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,872, filed Jan. 7, 2000, and 60/126,507, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07726, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,877, filed Jan. 7, 2000, and 60/126,597, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07677, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,064, filed Jan. 14, 2000, 60/154,373, filed Sep. 17, 1999, and 60/126,601, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/07725, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,063, filed Jan. 14, 2000, and 60/126,602, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/09070, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,052, filed Jan. 14, 2000, and 60/128,695, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/08982, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,069, filed Jan. 14, 2000, and 60/128,696, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/08983, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,068, filed Jan. 14, 2000, and 60/128,703, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/09067, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,929, filed Jan. 20, 2000, and 60/128,697, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/09066, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,926, filed Jan. 20, 2000, and 60/128,698, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/09068, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/177,050, filed Jan. 20, 2000, and 60/128,699, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/08981, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/177,166, filed Jan. 20, 2000, and 60/128,701, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/08980, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,930, filed Jan. 20, 2000, and 60/128,700, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/09071, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,931, filed Jan. 20, 2000, and 60/128,694, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/09069, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/177,049, filed Jan. 20, 2000, and 60/128,702, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/15136, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,629, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14926, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,628, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14963, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,631, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/15135, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,632, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14934, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,599, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14933, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,572, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/15137, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,625, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14928, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,633, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14973, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,630, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/14964, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,627, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/26376, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,808, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/26371, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,804, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/26324, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,807, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/26323, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,805, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US00/26337, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,806, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,082 is a continuation-in-part of International Application No. PCT/US01/13318, filed Apr. 26, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/212,142, filed Jun. 16, 2000, and 60/201,194, filed May 2, 2000; U.S. application Ser. No. 10/105,299 is a continuation-in-part of U.S. application Ser. No. 09/950,083, filed Sep. 12, 2001; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06043, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/167,061, filed Nov. 23, 1999, and 60/124,146, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06012, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/166,989, filed Nov. 23, 1999, and 60/124,093, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06058, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,654, filed Dec. 3, 1999, and 60/124,145, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06044, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,661, filed Dec. 3, 1999, and 60/124,099, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06059, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,622, filed Dec. 3, 1999, and 60/124,096, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06042, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,663, filed Dec. 3, 1999, and 60/124,143, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06014, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,665, filed Dec. 3, 1999, 60/138,598, filed Jun. 11, 1999, and 60/124,095, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06013, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,662, filed Dec. 3, 1999, 60/138,626, filed Jun. 11, 1999, and 60/125,360, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06049, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,667, filed Dec. 3, 1999, 60/138,574, filed Jun. 11, 1999, and 60/124,144, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06057, filed Mar. 9, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,666, filed Dec. 3, 1999, 60/138,597, filed Jun. 11, 1999, and 60/124,142, filed Mar. 12, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06824, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/168,664, filed Dec. 3, 1999, and 60/125,359, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06765, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,906, filed Dec. 10, 1999, and 60/126,051, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06792, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,980, filed Dec. 10, 1999, and 60/125,362, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06830, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,910, filed Dec. 10, 1999, and 60/125,361, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06782, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,936, filed Dec. 10, 1999, and 60/125,812, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06822, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,916, filed Dec. 10, 1999, and 60/126,054, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06791, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,946, filed Dec. 10, 1999, and 60/125,815, filed Mar. 23, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06828, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,616, filed Dec. 8, 1999, and 60/125,358, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06823, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,623, filed Dec. 8, 1999, and 60/125,364, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/06781, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/169,617, filed Dec. 8, 1999, and 60/125,363, filed Mar. 19, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07505, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,410, filed Dec. 17, 1999, and 60/126,502, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07440, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,409, filed Dec. 17, 1999, and 60/126,503, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07506, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,412, filed Dec. 17, 1999, and 60/126,505, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07507, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,408, filed Dec. 17, 1999, and 60/126,594, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07535, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/172,413, filed Dec. 17, 1999, and 60/126,511, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07525, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,549, filed Dec. 22, 1999, and 60/126,595, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07534, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,504, filed Dec. 22, 1999, and 60/126,598, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07483, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,552, filed Dec. 22, 1999, and 60/126,596, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07526, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,550, filed Dec. 22, 1999, and 60/126,600, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07527, filed Mar. 22, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/171,551, filed Dec. 22, 1999, and 60/126,501, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07661, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,847, filed Jan. 7, 2000, and 60/126,504, filed Mar. 26, 1999; U.S. Application No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07579, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,853, filed Jan. 7, 2000, and 60/126,509, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07723, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,852, filed Jan. 7, 2000, and 60/126,506, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07724, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,850, filed Jan. 7, 2000, and 60/126,510, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14929, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,851, filed Jan. 7, 2000, and 60/138,573, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07722, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,871, filed Jan. 7, 2000, and 60/126,508, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07578, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,872, filed Jan. 7, 2000, and 60/126,507, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07726, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/174,877, filed Jan. 7, 2000, and 60/126,597, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07677, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,064, filed Jan. 14, 2000, 60/154,373, filed Sep. 17, 1999, and 60/126,601, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/07725, filed Mar. 23, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,063, filed Jan. 14, 2000, and 60/126,602, filed Mar. 26, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/09070, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,052, filed Jan. 14, 2000, and 60/128,695, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/08982, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,069, filed Jan. 14, 2000, and 60/128,696, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/08983, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,068, filed Jan. 14, 2000, and 60/128,703, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/09067, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,929, filed Jan. 20, 2000, and 60/128,697, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/09066, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,926, filed Jan. 20, 2000, and 60/128,698, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/09068, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/177,050, filed Jan. 20, 2000, and 60/128,699, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/08981, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/177,166, filed Jan. 20, 2000, and 60/128,701, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/08980, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,930, filed Jan. 20, 2000, and 60/128,700, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/09071, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/176,931, filed Jan. 20, 2000, and 60/128,694, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/09069, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/177,049, filed Jan. 20, 2000, and 60/128,702, filed Apr. 9, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/15136, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,629, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14926, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,628, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14963, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,631, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/15135, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,632, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14934, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,599, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14933, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,572, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/15137, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,625, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14928, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,633, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14973, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,630, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/14964, filed Jun. 1, 2000, which claims the benefit of U.S. Provisional Application No. 60/138,627, filed Jun. 11, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/26376, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,808, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/26371, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,804, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/26324, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,807, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/26323, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,805, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US00/26337, filed Sep. 26, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,806, filed Sep. 27, 1999; U.S. application Ser. No. 09/950,083 is a continuation-in-part of International Application No. PCT/US01/13318, filed Apr. 26, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/212,142, filed Jun. 16, 2000, and 60/201,194, filed May 2, 2000. This application is a continuation-in-part of U.S. application Ser. No. 11/366,486, filed Mar. 3, 2006, which is a continuation-in-part of 10/664,358, filed Sep. 20, 2003, which is a continuation-in-part of International Application No. PCT/US02/09785, filed Sep. 20, 2003; International Application No. PCT/US02/09785 is a continuation-in-part of U.S. application Ser. No. 10/100,683, filed Mar. 19, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/277,340, filed Mar. 21, 2001, 60/306,171, filed Jul. 19, 2001, and 60/331,287, filed Nov. 13, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/981,876, filed Oct. 19, 2001, which is a divisional of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,161, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,615, filed May 23, 1997, 60/047,600, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/047,595, filed May 23, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, and 60/056,884, filed Aug. 22, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/882,171, filed Jun. 18, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/882,171 is a continuation of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/809,391 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,161, filed Mar. 7, 1997, 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,600, filed May 23, 1997, 60/047,615, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,595, filed May 23, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, 60/056,884, filed Aug. 22, 1997, 60/057,669, filed Sep. 5, 1997, 60/049,610, filed Jun. 13, 1997, 60/061,060, filed Oct. 2, 1997, 60/051,926, filed Jul. 8, 1997, 60/052,874, filed Jul. 16, 1997, 60/058,785, filed Sep. 12, 1997, and 60/055,724, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/058,993, filed Jan. 30, 2002, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,659 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/853,161 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,797 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,762, filed Mar. 14, 1997, 60/040,710, filed Mar. 14, 1997, 60/050,934, filed May 30, 1997, 60/048,100, filed May 30, 1997, 60/048,357, filed May 30, 1997, 60/048,189, filed May 30, 1997, 60/057,765, filed Sep. 5, 1997, 60/048,970, filed Jun. 6, 1997, and 60/068,368, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/059,395, filed Jan. 31, 2002, which is a divisional of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,245, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,966, filed Oct. 26, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/041,277, filed Mar. 21, 1997, 60/042,344, filed Mar. 21, 1997, 60/041,276, filed Mar. 21, 1997, 60/041,281, filed Mar. 21, 1997, 60/048,094, filed May 30, 1997, 60/048,350, filed May 30, 1997, 60/048,188, filed May 30, 1997, 60/048,135, filed May 30, 1997, 60/050,937, filed May 30, 1997, 60/048,187, filed May 30, 1997, 60/048,099, filed May 30, 1997, 60/048,352, filed May 30, 1997, 60/048,186, filed May 30, 1997, 60/048,069, filed May 30, 1997, 60/048,095, filed May 30, 1997, 60/048,131, filed May 30, 1997, 60/048,096, filed May 30, 1997, 60/048,355, filed May 30, 1997, 60/048,160, filed May 30, 1997, 60/048,351, filed May 30, 1997, 60/048,154, filed May 30, 1997, 60/054,804, filed Aug. 5, 1997, 60/056,370, filed Aug. 19, 1997, and 60/060,862, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/814,122, filed Mar. 22, 2001, which is a continuation of U.S. application Ser. No. 09/577,145, filed May 24, 2000, which is a continuation of U.S. application Ser. No. 09/166,780, filed Oct. 6, 1998, which is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/10868, filed May 28, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/044,039, filed May 30, 1997, 60/048,093, filed May 30, 1997, 60/048,190, filed May 30, 1997, 60/050,935, filed May 30, 1997, 60/048,101, filed May 30, 1997, 60/048,356, filed May 30, 1997, 60/056,250, filed Aug. 29, 1997, 60/056,296, filed Aug. 29, 1997, and 60/056,293, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/11422, filed Jun. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/048,885, filed Jun. 6, 1997, 60/049,375, filed Jun. 6, 1997, 60/048,881, filed Jun. 6, 1997, 60/048,880, filed Jun. 6, 1997, 60/048,896, filed Jun. 6, 1997, 60/049,020, filed Jun. 6, 1997, 60/048,876, filed Jun. 6, 1997, 60/048,895, filed Jun. 6, 1997, 60/048,884, filed Jun. 6, 1997, 60/048,894, filed Jun. 6, 1997, 60/048,971, filed Jun. 6, 1997, 60/048,964, filed Jun. 6, 1997, 60/048,882, filed Jun. 6, 1997, 60/048,899, filed Jun. 6, 1997, 60/048,893, filed Jun. 6, 1997, 60/048,900, filed Jun. 6, 1997, 60/048,901, filed Jun. 6, 1997, 60/048,892, filed Jun. 6, 1997, 60/048,915, filed Jun. 6, 1997, 60/049,019, filed Jun. 6, 1997, 60/048,970, filed Jun. 6, 1997, 60/048,972, filed Jun. 6, 1997, 60/048,916, filed Jun. 6, 1997, 60/049,373, filed Jun. 6, 1997, 60/048,875, filed Jun. 6, 1997, 60/049,374, filed Jun. 6, 1997, 60/048,917, filed Jun. 6, 1997, 60/048,949, filed Jun. 6, 1997, 60/048,974, filed Jun. 6, 1997, 60/048,883, filed Jun. 6, 1997, 60/048,897, filed Jun. 6, 1997, 60/048,898, filed Jun. 6, 1997, 60/048,962, filed Jun. 6, 1997, 60/048,963, filed Jun. 6, 1997, 60/048,877, filed Jun. 6, 1997, 60/048,878, filed Jun. 6, 1997, 60/057,645, filed Sep. 5, 1997, 60/057,642, filed Sep. 5, 1997, 60/057,668, filed Sep. 5, 1997, 60/057,635, filed Sep. 5, 1997, 60/057,627, filed Sep. 5, 1997, 60/057,667, filed Sep. 5, 1997, 60/057,666, filed Sep. 5, 1997, 60/057,764, filed Sep. 5, 1997, 60/057,643, filed Sep. 5, 1997, 60/057,769, filed Sep. 5, 1997, 60/057,763, filed Sep. 5, 1997, 60/057,650, filed Sep. 5, 1997, 60/057,584, filed Sep. 5, 1997, 60/057,647, filed Sep. 5, 1997, 60/057,661, filed Sep. 5, 1997, 60/057,662, filed Sep. 5, 1997, 60/057,646, filed Sep. 5, 1997, 60/057,654, filed Sep. 5, 1997, 60/057,651, filed Sep. 5, 1997, 60/057,644, filed Sep. 5, 1997, 60/057,765, filed Sep. 5, 1997, 60/057,762, filed Sep. 5, 1997, 60/057,775, filed Sep. 5, 1997, 60/057,648, filed Sep. 5, 1997, 60/057,774, filed Sep. 5, 1997, 60/057,649, filed Sep. 5, 1997, 60/057,770, filed Sep. 5, 1997, 60/057,771, filed Sep. 5, 1997, 60/057,761, filed Sep. 5, 1997, 60/057,760, filed Sep. 5, 1997, 60/057,776, filed Sep. 5, 1997, 60/057,778, filed Sep. 5, 1997, 60/057,629, filed Sep. 5, 1997, 60/057,628, filed Sep. 5, 1997, 60/057,777, filed Sep. 5, 1997, 60/057,634, filed Sep. 5, 1997, and 60/070,923, filed Dec. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/05614, filed Feb. 21, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/184,836, filed Feb. 24, 2000, and 60/193,170, filed Mar. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/12125, filed Jun. 11, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/049,547, filed Jun. 13, 1997, 60/049,548, filed Jun. 13, 1997, 60/049,549, filed Jun. 13, 1997, 60/049,550, filed Jun. 13, 1997, 60/049,566, filed Jun. 13, 1997, 60/049,606, filed Jun. 13, 1997, 60/049,607, filed Jun. 13, 1997, 60/049,608, filed Jun. 13, 1997, 60/049,609, filed Jun. 13, 1997, 60/049,610, filed Jun. 13, 1997, 60/049,611, filed Jun. 13, 1997, 60/050,901, filed Jun. 13, 1997, 60/052,989, filed Jun. 13, 1997, 60/051,919, filed Jul. 8, 1997, 60/055,984, filed Aug. 18, 1997, 60/058,665, filed Sep. 12, 1997, 60/058,668, filed Sep. 12, 1997, 60/058,669, filed Sep. 12, 1997, 60/058,750, filed Sep. 12, 1997, 60/058,971, filed Sep. 12, 1997, 60/058,972, filed Sep. 12, 1997, 60/058,975, filed Sep. 12, 1997, 60/060,834, filed Oct. 2, 1997, 60/060,841, filed Oct. 2, 1997, 60/060,844, filed Oct. 2, 1997, 60/060,865, filed Oct. 2, 1997, 60/061,059, filed Oct. 2, 1997, and 60/061,060, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/627,081, filed Jul. 27, 2000, which is a continuation of U.S. application Ser. No. 09/213,365, filed Dec. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,480, filed Jul. 1, 1997, 60/051,381, filed Jul. 1, 1997, 60/058,663, filed Sep. 12, 1997, and 60/058,598, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,490, filed Oct. 30, 2001, which is a divisional of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,802, filed Oct. 25, 2001, which is a continuation of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/973,278, filed Oct. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,899, filed Oct. 13, 2000; U.S. application Ser. No. 09/973,278 is a continuation-in-part of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,926, filed Jul. 8, 1997, 60/052,793, filed Jul. 8, 1997, 60/051,925, filed Jul. 8, 1997, 60/051,929, filed Jul. 8, 1997, 60/052,803, filed Jul. 8, 1997, 60/052,732, filed Jul. 8, 1997, 60/051,931, filed Jul. 8, 1997, 60/051,932, filed Jul. 8, 1997, 60/051,916, filed Jul. 8, 1997, 60/051,930, filed Jul. 8, 1997, 60/051,918, filed Jul. 8, 1997, 60/051,920, filed Jul. 8, 1997, 60/052,733, filed Jul. 8, 1997, 60/052,795, filed Jul. 8, 1997, 60/051,919, filed Jul. 8, 1997, 60/051,928, filed Jul. 8, 1997, 60/055,722, filed Aug. 18, 1997, 60/055,723, filed Aug. 18, 1997, 60/055,948, filed Aug. 18, 1997, 60/055,949, filed Aug. 18, 1997, 60/055,953, filed Aug. 18, 1997, 60/055,950, filed Aug. 18, 1997, 60/055,947, filed Aug. 18, 1997, 60/055,964, filed Aug. 18, 1997, 60/056,360, filed Aug. 18, 1997, 60/055,684, filed Aug. 18, 1997, 60/055,984, filed Aug. 18, 1997, 60/055,954, filed Aug. 18, 1997, 60/058,785, filed Sep. 12, 1997, 60/058,664, filed Sep. 12, 1997, 60/058,660, filed Sep. 12, 1997, and 60/058,661, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/776,724, filed Feb. 6, 2001, which claims the benefit of U.S. Provisional Application No. 60/180,909, filed Feb. 8, 2000; U.S. application Ser. No. 09/776,724 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/052,661, filed Jul. 16, 1997, 60/052,872, filed Jul. 16, 1997, 60/052,871, filed Jul. 16, 1997, 60/052,874, filed Jul. 16, 1997, 60/052,873, filed Jul. 16, 1997, 60/052,870, filed Jul. 16, 1997, 60/052,875, filed Jul. 16, 1997, 60/053,440, filed Jul. 22, 1997, 60/053,441, filed Jul. 22, 1997, 60/053,442, filed Jul. 22, 1997, 60/056,359, filed Aug. 18, 1997, 60/055,725, filed Aug. 18, 1997, 60/055,985, filed Aug. 18, 1997, 60/055,952, filed Aug. 18, 1997, 60/055,989, filed Aug. 18, 1997, 60/056,361, filed Aug. 18, 1997, 60/055,726, filed Aug. 18, 1997, 60/055,724, filed Aug. 18, 1997, 60/055,946, filed Aug. 18, 1997, and 60/055,683, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/295,558, filed Jun. 5, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,649, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/666,984, filed Sep. 21, 2000, which is a continuation of U.S. application Ser. No. 09/236,557, filed Jan. 26, 1999, which is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/054,212, filed Jul. 30, 1997, 60/054,209, filed Jul. 30, 1997, 60/054,234, filed Jul. 30, 1997, 60/054,218, filed Jul. 30, 1997, 60/054,214, filed Jul. 30, 1997, 60/054,236, filed Jul. 30, 1997, 60/054,215, filed Jul. 30, 1997, 60/054,211, filed Jul. 30, 1997, 60/054,217, filed Jul. 30, 1997, 60/054,213, filed Jul. 30, 1997, 60/055,968, filed Aug. 18, 1997, 60/055,969, filed Aug. 18, 1997, 60/055,972, filed Aug. 18, 1997, 60/056,561, filed Aug. 19, 1997, 60/056,534, filed Aug. 19, 1997, 60/056,729, filed Aug. 19, 1997, 60/056,543, filed Aug. 19, 1997, 60/056,727, filed Aug. 19, 1997, 60/056,554, filed Aug. 19, 1997, and 60/056,730, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which claims the benefit of U.S. Provisional Application No. 60/238,291, filed Oct. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/055,386, filed Aug. 5, 1997, 60/054,807, filed Aug. 5, 1997, 60/055,312, filed Aug. 5, 1997, 60/055,309, filed Aug. 5, 1997, 60/054,798, filed Aug. 5, 1997, 60/055,310, filed Aug. 5, 1997, 60/054,806, filed Aug. 5, 1997, 60/054,809, filed Aug. 5, 1997, 60/054,804, filed Aug. 5, 1997, 60/054,803, filed Aug. 5, 1997, 60/054,808, filed Aug. 5, 1997, 60/055,311, filed Aug. 5, 1997, 60/055,986, filed Aug. 18, 1997, 60/055,970, filed Aug. 18, 1997, 60/056,563, filed Aug. 19, 1997, 60/056,557, filed Aug. 19, 1997, 60/056,731, filed Aug. 19, 1997, 60/056,365, filed Aug. 19, 1997, 60/056,367, filed Aug. 19, 1997, 60/056,370, filed Aug. 19, 1997, 60/056,364, filed Aug. 19, 1997, 60/056,366, filed Aug. 19, 1997, 60/056,732, filed Aug. 19, 1997, and 60/056,371, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,128, filed Nov. 17, 2000, which is a continuation of U.S. application Ser. No. 09/251,329, filed Feb. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,555, filed Aug. 19, 1997, 60/056,556, filed Aug. 19, 1997, 60/056,535, filed Aug. 19, 1997, 60/056,629, filed Aug. 19, 1997, 60/056,369, filed Aug. 19, 1997, 60/056,628, filed Aug. 19, 1997, 60/056,728, filed Aug. 19, 1997, 60/056,368, filed Aug. 19, 1997, 60/056,726, filed Aug. 19, 1997, 60/089,510, filed Jun. 16, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/729,835, filed Dec. 6, 2000, which is a divisional of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,270, filed Aug. 29, 1997, 60/056,271, filed Aug. 29, 1997, 60/056,247, filed Aug. 29, 1997, and 60/056,073, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/047,021, filed Jan. 17, 2002, which is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/01109, filed Jan. 17, 2002, which claims the benefit of U.S. Provisional Application No. 60/262,066, filed Jan. 18, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/057,626, filed Sep. 5, 1997, 60/057,663, filed Sep. 5, 1997, 60/057,669, filed Sep. 5, 1997, 60/058,667, filed Sep. 12, 1997, 60/058,974, filed Sep. 12, 1997, 60/058,973, filed Sep. 12, 1997, 60/058,666, filed Sep. 12, 1997, and 60/090,112, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/281,976, filed Mar. 31, 1999, which is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998; U.S. Application No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/060,837, filed Oct. 2, 1997, 60/060,862, filed Oct. 2, 1997, 60/060,839, filed Oct. 2, 1997, 60/060,866, filed Oct. 2, 1997, 60/060,843, filed Oct. 2, 1997, 60/060,836, filed Oct. 2, 1997, 60/060,838, filed Oct. 2, 1997, 60/060,874, filed Oct. 2, 1997, 60/060,833, filed Oct. 2, 1997, 60/060,884, filed Oct. 2, 1997, and 60/060,880, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,429, filed Oct. 30, 2001, which claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 09/984,429 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/061,463, filed Oct. 9, 1997, 60/061,529, filed Oct. 9, 1997, 60/071,498, filed Oct. 9, 1997, 60/061,527, filed Oct. 9, 1997, 60/061,536, filed Oct. 9, 1997, and 60/061,532, filed Oct. 9, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/296,622, filed Apr. 23, 1999, which is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/063,099, filed Oct. 24, 1997, 60/063,088, filed Oct. 24, 1997, 60/063,100, filed Oct. 24, 1997, 60/063,387, filed Oct. 24, 1997, 60/063,148, filed Oct. 24, 1997, 60/063,386, filed Oct. 24, 1997, 60/062,784, filed Oct. 24, 1997, 60/063,091, filed Oct. 24, 1997, 60/063,090, filed Oct. 24, 1997, 60/063,089, filed Oct. 24, 1997, 60/063,092, filed Oct. 24, 1997, 60/063,111, filed Oct. 24, 1997, 60/063,101, filed Oct. 24, 1997, 60/063,109, filed Oct. 24, 1997, 60/063,110, filed Oct. 24, 1997, 60/063,098, filed Oct. 24, 1997, and 60/063,097, filed Oct. 24, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/974,879, filed Oct. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,893, filed Oct. 13, 2000; U.S. application Ser. No. 09/974,879 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/064,911, filed Nov. 7, 1997, 60/064,912, filed Nov. 7, 1997, 60/064,983, filed Nov. 7, 1997, 60/064,900, filed Nov. 7, 1997, 60/064,988, filed Nov. 7, 1997, 60/064,987, filed Nov. 7, 1997, 60/064,908, filed Nov. 7, 1997, 60/064,984, filed Nov. 7, 1997, 60/064,985, filed Nov. 7, 1997, 60/066,094, filed Nov. 17, 1997, 60/066,100, filed Nov. 17, 1997, 60/066,089, filed Nov. 17, 1997, 60/066,095, filed Nov. 17, 1997, and 60/066,090, filed Nov. 17, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/334,595, filed Jun. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/070,923, filed Dec. 18, 1997, 60/068,007, filed Dec. 18, 1997, 60/068,057, filed Dec. 18, 1997, 60/068,006, filed Dec. 18, 1997, 60/068,369, filed Dec. 19, 1997, 60/068,367, filed Dec. 19, 1997, 60/068,368, filed Dec. 19, 1997, 60/068,169, filed Dec. 19, 1997, 60/068,053, filed Dec. 18, 1997, 60/068,064, filed Dec. 18, 1997, 60/068,054, filed Dec. 18, 1997, 60/068,008, filed Dec. 18, 1997, and 60/068,365, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/938,671, filed Aug. 27, 2001, which is a continuation of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/070,704, filed Jan. 7, 1998, 60/070,658, filed Jan. 7, 1998, 60/070,692, filed Jan. 7, 1998, and 60/070,657, filed Jan. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/949,925, filed Sep. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,150, filed Sep. 12, 2000; U.S. application Ser. No. 09/949,925 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 09/949,925 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/813,153, filed Mar. 21, 2001, which is a continuation of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/073,170, filed Jan. 30, 1998, 60/073,167, filed Jan. 30, 1998, 60/073,165, filed Jan. 30, 1998, 60/073,164, filed Jan. 30, 1998, 60/073,162, filed Jan. 30, 1998, 60/073,161, filed Jan. 30, 1998, 60/073,160, filed Jan. 30, 1998, and 60/073,159, filed Jan. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/062,548, filed Feb. 5, 2002, which is a continuation of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/074,118, filed Feb. 9, 1998, 60/074,157, filed Feb. 9, 1998, 60/074,037, filed Feb. 9, 1998, 60/074,141, filed Feb. 9, 1998, and 60/074,341, filed Feb. 9, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,129, filed Nov. 17, 2000, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 09/716,129 is a continuation of U.S. application Ser. No. 09/382,572, filed Aug. 25, 1999, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/076,053, filed Feb. 26, 1998, 60/076,051, filed Feb. 26, 1998, 60/076,054, filed Feb. 26, 1998, 60/076,052, filed Feb. 26, 1998, and 60/076,057, filed Feb. 26, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/798,889, filed Mar. 6, 2001, which is a continuation of U.S. application Ser. No. 09/393,022, filed Sep. 9, 1999, which is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/077,714, filed Mar. 12, 1998, 60/077,686, filed Mar. 12, 1998, 60/077,687, filed Mar. 12, 1998, and 60/077,696, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/397,945, filed Sep. 17, 1999, which is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/078,566, filed Mar. 19, 1998, 60/078,576, filed Mar. 19, 1998, 60/078,573, filed Mar. 19, 1998, 60/078,574, filed Mar. 19, 1998, 60/078,579, filed Mar. 19, 1998, 60/080,314, filed Apr. 1, 1998, 60/080,312, filed Apr. 1, 1998, 60/078,578, filed Mar. 19, 1998, 60/078,581, filed Mar. 19, 1998, 60/078,577, filed Mar. 19, 1998, 60/078,563, filed Mar. 19, 1998, and 60/080,313, filed Apr. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,783, filed Sep. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,846, filed Sep. 11, 2000; U.S. Application No. 09/948,783 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/085,093, filed May 12, 1998, 60/085,094, filed May 12, 1998, 60/085,105, filed May 12, 1998, 60/085,180, filed May 12, 1998, 60/085,927, filed May 18, 1998, 60/085,906, filed May 18, 1998, 60/085,920, filed May 18, 1998, 60/085,924, filed May 18, 1998, 60/085,922, filed May 18, 1998, 60/085,923, filed May 18, 1998, 60/085,921, filed May 18, 1998, 60/085,925, filed May 18, 1998, and 60/085,928, filed May 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,873, filed Jan. 18, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/263,681, filed Jan. 24, 2001, and 60/263,230, filed Jan. 23, 2001; U.S. application Ser. No. 10/050,873 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/012,542, filed Dec. 12, 2001, which is a divisional of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/089,507, filed Jun. 16, 1998, 60/089,508, filed Jun. 16, 1998, 60/089,509, filed Jun. 16, 1998, 60/089,510, filed Jun. 16, 1998, 60/090,112, filed Jun. 22, 1998, and 60/090,113, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,271, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,276, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/092,921, filed Jul. 15, 1998, 60/092,922, filed Jul. 15, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/29871, filed Sep. 24, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,925, filed Sep. 25, 2000; International Application No. PCT/US01/29871 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/350,898, filed Jan. 25, 2002; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/489,847, filed Jan. 24, 2000, which is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/094,657, filed Jul. 30, 1998, 60/095,486, filed Aug. 5, 1998, 60/096,319, filed Aug. 12, 1998, 60/095,454, filed Aug. 6, 1998, and 60/095,455, filed Aug. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/054,988, filed Jan. 25, 2002, which is a continuation of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. Application No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/097,917, filed Aug. 25, 1998, and 60/098,634, filed Aug. 31, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,893, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/531,119, filed Mar. 20, 2000, which is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/101,546, filed Sep. 23, 1998, and 60/102,895, filed Oct. 2, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,820, filed Sep. 10, 2001, which is a continuation of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999, which claims the benefit of U.S. Provisional Application No. 60/108,207, filed Nov. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/895,298, filed Jul. 2, 2001, which is a continuation of U.S. application Ser. No. 09/591,316, filed Jun. 9, 2000, which is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/113,006, filed Dec. 18, 1998, and 60/112,809, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/985,153, filed Nov. 1, 2001, which is a continuation of U.S. application Ser. No. 09/618,150, filed Jul. 17, 2000, which is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000, which claims the benefit of U.S. Provisional Application No. 60/116,330, filed Jan. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/997,131, filed Nov. 30, 2001, which is a continuation of U.S. application Ser. No. 09/628,508, filed Jul. 28, 2000, which is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000, which claims the benefit of U.S. Provisional Application No. 60/119,468, filed Feb. 10, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,882, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/125,055, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,704, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/128,693, filed Apr. 9, 1999, and 60/130,991, filed Apr. 26, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/042,141, filed Jan. 11, 2002, which is a continuation of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000, which claims the benefit of U.S. Provisional Application No. 60/137,725, filed Jun. 7, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/756,168, filed Jan. 9, 2001, which is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 20, 2000, which claims the benefit of U.S. Provisional Application No. 60/145,220, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/060,255, filed Feb. 1, 2002, which is a continuation of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/149,182, filed Aug. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/789,561, filed Feb. 22, 2001, which is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/152,315, filed Sep. 3, 1999, and 60/152,317, filed Sep. 3, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/800,729, filed Mar. 8, 2001, which is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,709, filed Sep. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/832,129, filed Apr. 11, 2001, which is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/163,085, filed Nov. 2, 1999, and 60/172,411, filed Dec. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29363, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,139, filed Jun. 30, 2000, and 60/162,239, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29360, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,138, filed Jun. 30, 2000, and 60/162,211, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29362, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,131, filed Jun. 30, 2000, and 60/162,240, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29365, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,666, filed Jul. 21, 2000, and 60/162,237, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29364, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,134, filed Jun. 30, 2000, and 60/162,238, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30040, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,130, filed Jun. 30, 2000, and 60/163,580, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30037, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,137, filed Jun. 30, 2000, and 60/163,577, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30045, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,133, filed Jun. 30, 2000, and 60/163,581, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30036, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,366, filed Jul. 27, 2000, and 60/163,576, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30039, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,367, filed Jul. 27, 2000, 60/195,296, filed Apr. 7, 2000, and 60/164,344, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30654, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,142, filed Jul. 27, 2000, and 60/164,835, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30628, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,140, filed Jun. 30, 2000, and 60/164,744, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30653, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,193, filed Jul. 27, 2000, and 60/164,735, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30629, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/222,904, filed Aug. 3, 2000, and 60/164,825, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30679, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/224,007, filed Aug. 4, 2000, and 60/164,834, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30674, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,128, filed Jun. 30, 2000, and 60/164,750, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31162, filed Nov. 15, 2000; International Application No. PCT/US00/31162, filed Nov. 15, 2000 claims the benefit of U.S. Provisional Application Nos. 60/215,136, filed Jun. 30, 2000, and 60/166,415, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31282, filed Nov. 15, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,665, filed Jul. 21, 2000, and 60/166,414, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30657, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,132, filed Jun. 30, 2000, and 60/164,731, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01396, filed Jan. 17, 2001; International Application No. PCT/US01/01396, filed Jan. 17, 2001 claims the benefit of U.S. Provisional Application Nos. 60/256,968, filed Dec. 21, 2000, and 60/226,280, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01387, filed Jan. 17, 2001; International Application No. PCT/US01/01387, filed Jan. 17, 2001 claims the benefit of U.S. Provisional Application Nos. 60/259,803, filed Jan. 5, 2001, and 60/226,380, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01567, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/228,084, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01431, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,968, filed Sep. 12, 2000; International Application No. PCT/US01/01431 is a continuation-in-part of U.S. Application No. 09/915,582, filed Jul. 27, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01432, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/236,326, filed Sep. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00544, filed Jan. 9, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,211, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01435, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,282, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01386, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,104, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01565, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,210, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01394, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,805, filed Jan. 5, 2001, and 60/226,278, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01434, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,678, filed Jan. 5, 2001, and 60/226,279, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01397, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,281, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01385, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,969, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01384, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,516, filed Jan. 4, 2001, and 60/228,086, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01383, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,804, filed Jan. 5, 2001, and 60/228,083, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05064, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,444, filed Jul. 12, 2001, and 60/270,658, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05301, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,417, filed Jul. 12, 2001, and 60/270,625, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application Nos. 60/304,121, filed Jul. 11, 2001, 60/295,869, filed Jun. 6, 2001, 60/325,209, filed Sep. 28, 2001, 60/311,085, filed Aug. 10, 2001, 60/330,629, filed Oct. 26, 2001, 60/331,046, filed Nov. 7, 2001, 60/358,554, filed Feb. 22, 2002, and 60/358,714, filed Feb. 25, 2002. This application is a continuation-in-part of U.S. application Ser. No. 11/687,755, filed Mar. 19, 2007, which is a divisional of U.S. patent application Ser. No. 10/664,356, filed Sep. 20, 2003, which is a continuation-in-part of PCT/US02/08123, filed Mar. 19, 2002, which is a continuation-in-part of U.S. application Ser. No. 10/100,683, filed Mar. 19, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/277,340, filed Mar. 21, 2001, 60/306,171, filed Jul. 19, 2001, and 60/331,287, filed Nov. 13, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/981,876, filed Oct. 19, 2001, which is a divisional of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,161, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,615, filed May 23, 1997, 60/047,600, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/047,595, filed May 23, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, and 60/056,884, filed Aug. 22, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/882,171, filed Jun. 18, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/882,171 is a continuation of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/809,391 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,161, filed Mar. 7, 1997, 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,600, filed May 23, 1997, 60/047,615, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,595, filed May 23, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, 60/056,884, filed Aug. 22, 1997, 60/057,669, filed Sep. 5, 1997, 60/049,610, filed Jun. 13, 1997, 60/061,060, filed Oct. 2, 1997, 60/051,926, filed Jul. 8, 1997, 60/052,874, filed Jul. 16, 1997, 60/058,785, filed Sep. 12, 1997, and 60/055,724, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/058,993, filed Jan. 30, 2002, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,659 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/853,161 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,797 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,762, filed Mar. 14, 1997, 60/040,710, filed Mar. 14, 1997, 60/050,934, filed May 30, 1997, 60/048,100, filed May 30, 1997, 60/048,357, filed May 30, 1997, 60/048,189, filed May 30, 1997, 60/057,765, filed Sep. 5, 1997, 60/048,970, filed Jun. 6, 1997, and 60/068,368, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/059,395, filed Jan. 31, 2002, which is a divisional of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,245, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,966, filed Oct. 26, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/041,277, filed Mar. 21, 1997, 60/042,344, filed Mar. 21, 1997, 60/041,276, filed Mar. 21, 1997, 60/041,281, filed Mar. 21, 1997, 60/048,094, filed May 30, 1997, 60/048,350, filed May 30, 1997, 60/048,188, filed May 30, 1997, 60/048,135, filed May 30, 1997, 60/050,937, filed May 30, 1997, 60/048,187, filed May 30, 1997, 60/048,099, filed May 30, 1997, 60/048,352, filed May 30, 1997, 60/048,186, filed May 30, 1997, 60/048,069, filed May 30, 1997, 60/048,095, filed May 30, 1997, 60/048,131, filed May 30, 1997, 60/048,096, filed May 30, 1997, 60/048,355, filed May 30, 1997, 60/048,160, filed May 30, 1997, 60/048,351, filed May 30, 1997, 60/048,154, filed May 30, 1997, 60/054,804, filed Aug. 5, 1997, 60/056,370, filed Aug. 19, 1997, and 60/060,862, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/814,122, filed Mar. 22, 2001, which is a continuation of U.S. application Ser. No. 09/577,145, filed May 24, 2000, which is a continuation of U.S. application Ser. No. 09/166,780, filed Oct. 6, 1998, which is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/10868, filed May 28, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/044,039, filed May 30, 1997, 60/048,093, filed May 30, 1997, 60/048,190, filed May 30, 1997, 60/050,935, filed May 30, 1997, 60/048,101, filed May 30, 1997, 60/048,356, filed May 30, 1997, 60/056,250, filed Aug. 29, 1997, 60/056,296, filed Aug. 29, 1997, and 60/056,293, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/11422, filed Jun. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/048,885, filed Jun. 6, 1997, 60/049,375, filed Jun. 6, 1997, 60/048,881, filed Jun. 6, 1997, 60/048,880, filed Jun. 6, 1997, 60/048,896, filed Jun. 6, 1997, 60/049,020, filed Jun. 6, 1997, 60/048,876, filed Jun. 6, 1997, 60/048,895, filed Jun. 6, 1997, 60/048,884, filed Jun. 6, 1997, 60/048,894, filed Jun. 6, 1997, 60/048,971, filed Jun. 6, 1997, 60/048,964, filed Jun. 6, 1997, 60/048,882, filed Jun. 6, 1997, 60/048,899, filed Jun. 6, 1997, 60/048,893, filed Jun. 6, 1997, 60/048,900, filed Jun. 6, 1997, 60/048,901, filed Jun. 6, 1997, 60/048,892, filed Jun. 6, 1997, 60/048,915, filed Jun. 6, 1997, 60/049,019, filed Jun. 6, 1997, 60/048,970, filed Jun. 6, 1997, 60/048,972, filed Jun. 6, 1997, 60/048,916, filed Jun. 6, 1997, 60/049,373, filed Jun. 6, 1997, 60/048,875, filed Jun. 6, 1997, 60/049,374, filed Jun. 6, 1997, 60/048,917, filed Jun. 6, 1997, 60/048,949, filed Jun. 6, 1997, 60/048,974, filed Jun. 6, 1997, 60/048,883, filed Jun. 6, 1997, 60/048,897, filed Jun. 6, 1997, 60/048,898, filed Jun. 6, 1997, 60/048,962, filed Jun. 6, 1997, 60/048,963, filed Jun. 6, 1997, 60/048,877, filed Jun. 6, 1997, 60/048,878, filed Jun. 6, 1997, 60/057,645, filed Sep. 5, 1997, 60/057,642, filed Sep. 5, 1997, 60/057,668, filed Sep. 5, 1997, 60/057,635, filed Sep. 5, 1997, 60/057,627, filed Sep. 5, 1997, 60/057,667, filed Sep. 5, 1997, 60/057,666, filed Sep. 5, 1997, 60/057,764, filed Sep. 5, 1997, 60/057,643, filed Sep. 5, 1997, 60/057,769, filed Sep. 5, 1997, 60/057,763, filed Sep. 5, 1997, 60/057,650, filed Sep. 5, 1997, 60/057,584, filed Sep. 5, 1997, 60/057,647, filed Sep. 5, 1997, 60/057,661, filed Sep. 5, 1997, 60/057,662, filed Sep. 5, 1997, 60/057,646, filed Sep. 5, 1997, 60/057,654, filed Sep. 5, 1997, 60/057,651, filed Sep. 5, 1997, 60/057,644, filed Sep. 5, 1997, 60/057,765, filed Sep. 5, 1997, 60/057,762, filed Sep. 5, 1997, 60/057,775, filed Sep. 5, 1997, 60/057,648, filed Sep. 5, 1997, 60/057,774, filed Sep. 5, 1997, 60/057,649, filed Sep. 5, 1997, 60/057,770, filed Sep. 5, 1997, 60/057,771, filed Sep. 5, 1997, 60/057,761, filed Sep. 5, 1997, 60/057,760, filed Sep. 5, 1997, 60/057,776, filed Sep. 5, 1997, 60/057,778, filed Sep. 5, 1997, 60/057,629, filed Sep. 5, 1997, 60/057,628, filed Sep. 5, 1997, 60/057,777, filed Sep. 5, 1997, 60/057,634, filed Sep. 5, 1997, and 60/070,923, filed Dec. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/05614, filed Feb. 21, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/184,836, filed Feb. 24, 2000, and 60/193,170, filed Mar. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/12125, filed Jun. 11, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/049,547, filed Jun. 13, 1997, 60/049,548, filed Jun. 13, 1997, 60/049,549, filed Jun. 13, 1997, 60/049,550, filed Jun. 13, 1997, 60/049,566, filed Jun. 13, 1997, 60/049,606, filed Jun. 13, 1997, 60/049,607, filed Jun. 13, 1997, 60/049,608, filed Jun. 13, 1997, 60/049,609, filed Jun. 13, 1997, 60/049,610, filed Jun. 13, 1997, 60/049,611, filed Jun. 13, 1997, 60/050,901, filed Jun. 13, 1997, 60/052,989, filed Jun. 13, 1997, 60/051,919, filed Jul. 8, 1997, 60/055,984, filed Aug. 18, 1997, 60/058,665, filed Sep. 12, 1997, 60/058,668, filed Sep. 12, 1997, 60/058,669, filed Sep. 12, 1997, 60/058,750, filed Sep. 12, 1997, 60/058,971, filed Sep. 12, 1997, 60/058,972, filed Sep. 12, 1997, 60/058,975, filed Sep. 12, 1997, 60/060,834, filed Oct. 2, 1997, 60/060,841, filed Oct. 2, 1997, 60/060,844, filed Oct. 2, 1997, 60/060,865, filed Oct. 2, 1997, 60/061,059, filed Oct. 2, 1997, and 60/061,060, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/627,081, filed Jul. 27, 2000, which is a continuation of U.S. application Ser. No. 09/213,365, filed Dec. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,480, filed Jul. 1, 1997, 60/051,381, filed Jul. 1, 1997, 60/058,663, filed Sep. 12, 1997, and 60/058,598, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,490, filed Oct. 30, 2001, which is a divisional of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,802, filed Oct. 25, 2001, which is a continuation of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/973,278, filed Oct. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,899, filed Oct. 13, 2000; U.S. application Ser. No. 09/973,278 is a continuation-in-part of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,926, filed Jul. 8, 1997, 60/052,793, filed Jul. 8, 1997, 60/051,925, filed Jul. 8, 1997, 60/051,929, filed Jul. 8, 1997, 60/052,803, filed Jul. 8, 1997, 60/052,732, filed Jul. 8, 1997, 60/051,931, filed Jul. 8, 1997, 60/051,932, filed Jul. 8, 1997, 60/051,916, filed Jul. 8, 1997, 60/051,930, filed Jul. 8, 1997, 60/051,918, filed Jul. 8, 1997, 60/051,920, filed Jul. 8, 1997, 60/052,733, filed Jul. 8, 1997, 60/052,795, filed Jul. 8, 1997, 60/051,919, filed Jul. 8, 1997, 60/051,928, filed Jul. 8, 1997, 60/055,722, filed Aug. 18, 1997, 60/055,723, filed Aug. 18, 1997, 60/055,948, filed Aug. 18, 1997, 60/055,949, filed Aug. 18, 1997, 60/055,953, filed Aug. 18, 1997, 60/055,950, filed Aug. 18, 1997, 60/055,947, filed Aug. 18, 1997, 60/055,964, filed Aug. 18, 1997, 60/056,360, filed Aug. 18, 1997, 60/055,684, filed Aug. 18, 1997, 60/055,984, filed Aug. 18, 1997, 60/055,954, filed Aug. 18, 1997, 60/058,785, filed Sep. 12, 1997, 60/058,664, filed Sep. 12, 1997, 60/058,660, filed Sep. 12, 1997, and 60/058,661, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/776,724, filed Feb. 6, 2001, which claims the benefit of U.S. Provisional Application No. 60/180,909, filed Feb. 8, 2000; U.S. application Ser. No. 09/776,724 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/052,661, filed Jul. 16, 1997, 60/052,872, filed Jul. 16, 1997, 60/052,871, filed Jul. 16, 1997, 60/052,874, filed Jul. 16, 1997, 60/052,873, filed Jul. 16, 1997, 60/052,870, filed Jul. 16, 1997, 60/052,875, filed Jul. 16, 1997, 60/053,440, filed Jul. 22, 1997, 60/053,441, filed Jul. 22, 1997, 60/053,442, filed Jul. 22, 1997, 60/056,359, filed Aug. 18, 1997, 60/055,725, filed Aug. 18, 1997, 60/055,985, filed Aug. 18, 1997, 60/055,952, filed Aug. 18, 1997, 60/055,989, filed Aug. 18, 1997, 60/056,361, filed Aug. 18, 1997, 60/055,726, filed Aug. 18, 1997, 60/055,724, filed Aug. 18, 1997, 60/055,946, filed Aug. 18, 1997, and 60/055,683, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/295,558, filed Jun. 5, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,649, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/666,984, filed Sep. 21, 2000, which is a continuation of U.S. application Ser. No. 09/236,557, filed Jan. 26, 1999, which is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/054,212, filed Jul. 30, 1997, 60/054,209, filed Jul. 30, 1997, 60/054,234, filed Jul. 30, 1997, 60/054,218, filed Jul. 30, 1997, 60/054,214, filed Jul. 30, 1997, 60/054,236, filed Jul. 30, 1997, 60/054,215, filed Jul. 30, 1997, 60/054,211, filed Jul. 30, 1997, 60/054,217, filed Jul. 30, 1997, 60/054,213, filed Jul. 30, 1997, 60/055,968, filed Aug. 18, 1997, 60/055,969, filed Aug. 18, 1997, 60/055,972, filed Aug. 18, 1997, 60/056,561, filed Aug. 19, 1997, 60/056,534, filed Aug. 19, 1997, 60/056,729, filed Aug. 19, 1997, 60/056,543, filed Aug. 19, 1997, 60/056,727, filed Aug. 19, 1997, 60/056,554, filed Aug. 19, 1997, and 60/056,730, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which claims the benefit of U.S. Provisional Application No. 60/238,291, filed Oct. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/055,386, filed Aug. 5, 1997, 60/054,807, filed Aug. 5, 1997, 60/055,312, filed Aug. 5, 1997, 60/055,309, filed Aug. 5, 1997, 60/054,798, filed Aug. 5, 1997, 60/055,310, filed Aug. 5, 1997, 60/054,806, filed Aug. 5, 1997, 60/054,809, filed Aug. 5, 1997, 60/054,804, filed Aug. 5, 1997, 60/054,803, filed Aug. 5, 1997, 60/054,808, filed Aug. 5, 1997, 60/055,311, filed Aug. 5, 1997, 60/055,986, filed Aug. 18, 1997, 60/055,970, filed Aug. 18, 1997, 60/056,563, filed Aug. 19, 1997, 60/056,557, filed Aug. 19, 1997, 60/056,731, filed Aug. 19, 1997, 60/056,365, filed Aug. 19, 1997, 60/056,367, filed Aug. 19, 1997, 60/056,370, filed Aug. 19, 1997, 60/056,364, filed Aug. 19, 1997, 60/056,366, filed Aug. 19, 1997, 60/056,732, filed Aug. 19, 1997, and 60/056,371, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,128, filed Nov. 17, 2000, which is a continuation of U.S. application Ser. No. 09/251,329, filed Feb. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,555, filed Aug. 19, 1997, 60/056,556, filed Aug. 19, 1997, 60/056,535, filed Aug. 19, 1997, 60/056,629, filed Aug. 19, 1997, 60/056,369, filed Aug. 19, 1997, 60/056,628, filed Aug. 19, 1997, 60/056,728, filed Aug. 19, 1997, 60/056,368, filed Aug. 19, 1997, 60/056,726, filed Aug. 19, 1997, 60/089,510, filed Jun. 16, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/729,835, filed Dec. 6, 2000, which is a divisional of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,270, filed Aug. 29, 1997, 60/056,271, filed Aug. 29, 1997, 60/056,247, filed Aug. 29, 1997, and 60/056,073, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/047,021, filed Jan. 17, 2002, which is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/01109, filed Jan. 17, 2002, which claims the benefit of U.S. Provisional Application No. 60/262,066, filed Jan. 18, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/057,626, filed Sep. 5, 1997, 60/057,663, filed Sep. 5, 1997, 60/057,669, filed Sep. 5, 1997, 60/058,667, filed Sep. 12, 1997, 60/058,974, filed Sep. 12, 1997, 60/058,973, filed Sep. 12, 1997, 60/058,666, filed Sep. 12, 1997, and 60/090,112, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/281,976, filed Mar. 31, 1999, which is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/060,837, filed Oct. 2, 1997, 60/060,862, filed Oct. 2, 1997, 60/060,839, filed Oct. 2, 1997, 60/060,866, filed Oct. 2, 1997, 60/060,843, filed Oct. 2, 1997, 60/060,836, filed Oct. 2, 1997, 60/060,838, filed Oct. 2, 1997, 60/060,874, filed Oct. 2, 1997, 60/060,833, filed Oct. 2, 1997, 60/060,884, filed Oct. 2, 1997, and 60/060,880, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,429, filed Oct. 30, 2001, which claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 09/984,429 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/061,463, filed Oct. 9, 1997, 60/061,529, filed Oct. 9, 1997, 60/071,498, filed Oct. 9, 1997, 60/061,527, filed Oct. 9, 1997, 60/061,536, filed Oct. 9, 1997, and 60/061,532, filed Oct. 9, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/296,622, filed Apr. 23, 1999, which is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/063,099, filed Oct. 24, 1997, 60/063,088, filed Oct. 24, 1997, 60/063,100, filed Oct. 24, 1997, 60/063,387, filed Oct. 24, 1997, 60/063,148, filed Oct. 24, 1997, 60/063,386, filed Oct. 24, 1997, 60/062,784, filed Oct. 24, 1997, 60/063,091, filed Oct. 24, 1997, 60/063,090, filed Oct. 24, 1997, 60/063,089, filed Oct. 24, 1997, 60/063,092, filed Oct. 24, 1997, 60/063,111, filed Oct. 24, 1997, 60/063,101, filed Oct. 24, 1997, 60/063,109, filed Oct. 24, 1997, 60/063,110, filed Oct. 24, 1997, 60/063,098, filed Oct. 24, 1997, and 60/063,097, filed Oct. 24, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/974,879, filed Oct. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,893, filed Oct. 13, 2000; U.S. application Ser. No. 09/974,879 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/064,911, filed Nov. 7, 1997, 60/064,912, filed Nov. 7, 1997, 60/064,983, filed Nov. 7, 1997, 60/064,900, filed Nov. 7, 1997, 60/064,988, filed Nov. 7, 1997, 60/064,987, filed Nov. 7, 1997, 60/064,908, filed Nov. 7, 1997, 60/064,984, filed Nov. 7, 1997, 60/064,985, filed Nov. 7, 1997, 60/066,094, filed Nov. 17, 1997, 60/066,100, filed Nov. 17, 1997, 60/066,089, filed Nov. 17, 1997, 60/066,095, filed Nov. 17, 1997, and 60/066,090, filed Nov. 17, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/334,595, filed Jun. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/070,923, filed Dec. 18, 1997, 60/068,007, filed Dec. 18, 1997, 60/068,057, filed Dec. 18, 1997, 60/068,006, filed Dec. 18, 1997, 60/068,369, filed Dec. 19, 1997, 60/068,367, filed Dec. 19, 1997, 60/068,368, filed Dec. 19, 1997, 60/068,169, filed Dec. 19, 1997, 60/068,053, filed Dec. 18, 1997, 60/068,064, filed Dec. 18, 1997, 60/068,054, filed Dec. 18, 1997, 60/068,008, filed Dec. 18, 1997, and 60/068,365, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/938,671, filed Aug. 27, 2001, which is a continuation of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/070,704, filed Jan. 7, 1998, 60/070,658, filed Jan. 7, 1998, 60/070,692, filed Jan. 7, 1998, and 60/070,657, filed Jan. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/949,925, filed Sep. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,150, filed Sep. 12, 2000; U.S. application Ser. No. 09/949,925 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 09/949,925 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/813,153, filed Mar. 21, 2001, which is a continuation of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/073,170, filed Jan. 30, 1998, 60/073,167, filed Jan. 30, 1998, 60/073,165, filed Jan. 30, 1998, 60/073,164, filed Jan. 30, 1998, 60/073,162, filed Jan. 30, 1998, 60/073,161, filed Jan. 30, 1998, 60/073,160, filed Jan. 30, 1998, and 60/073,159, filed Jan. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/062,548, filed Feb. 5, 2002, which is a continuation of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/074,118, filed Feb. 9, 1998, 60/074,157, filed Feb. 9, 1998, 60/074,037, filed Feb. 9, 1998, 60/074,141, filed Feb. 9, 1998, and 60/074,341, filed Feb. 9, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,129, filed Nov. 17, 2000, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 09/716,129 is a continuation of U.S. application Ser. No. 09/382,572, filed Aug. 25, 1999, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/076,053, filed Feb. 26, 1998, 60/076,051, filed Feb. 26, 1998, 60/076,054, filed Feb. 26, 1998, 60/076,052, filed Feb. 26, 1998, and 60/076,057, filed Feb. 26, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/798,889, filed Mar. 6, 2001, which is a continuation of U.S. application Ser. No. 09/393,022, filed Sep. 9, 1999, which is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/077,714, filed Mar. 12, 1998, 60/077,686, filed Mar. 12, 1998, 60/077,687, filed Mar. 12, 1998, and 60/077,696, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/397,945, filed Sep. 17, 1999, which is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/078,566, filed Mar. 19, 1998, 60/078,576, filed Mar. 19, 1998, 60/078,573, filed Mar. 19, 1998, 60/078,574, filed Mar. 19, 1998, 60/078,579, filed Mar. 19, 1998, 60/080,314, filed Apr. 1, 1998, 60/080,312, filed Apr. 1, 1998, 60/078,578, filed Mar. 19, 1998, 60/078,581, filed Mar. 19, 1998, 60/078,577, filed Mar. 19, 1998, 60/078,563, filed Mar. 19, 1998, and 60/080,313, filed Apr. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,783, filed Sep. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,846, filed Sep. 11, 2000; U.S. application Ser. No. 09/948,783 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/085,093, filed May 12, 1998, 60/085,094, filed May 12, 1998, 60/085,105, filed May 12, 1998, 60/085,180, filed May 12, 1998, 60/085,927, filed May 18, 1998, 60/085,906, filed May 18, 1998, 60/085,920, filed May 18, 1998, 60/085,924, filed May 18, 1998, 60/085,922, filed May 18, 1998, 60/085,923, filed May 18, 1998, 60/085,921, filed May 18, 1998, 60/085,925, filed May 18, 1998, and 60/085,928, filed May 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,873, filed Jan. 18, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/263,681, filed Jan. 24, 2001, and 60/263,230, filed Jan. 23, 2001; U.S. application Ser. No. 10/050,873 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/012,542, filed Dec. 12, 2001, which is a divisional of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/089,507, filed Jun. 16, 1998, 60/089,508, filed Jun. 16, 1998, 60/089,509, filed Jun. 16, 1998, 60/089,510, filed Jun. 16, 1998, 60/090,112, filed Jun. 22, 1998, and 60/090,113, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,271, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,276, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/092,921, filed Jul. 15, 1998, 60/092,922, filed Jul. 15, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/29871, filed Sep. 24, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,925, filed Sep. 25, 2000; International Application No. PCT/US01/29871 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/350,898, filed Jan. 25, 2002; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/489,847, filed Jan. 24, 2000, which is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/094,657, filed Jul. 30, 1998, 60/095,486, filed Aug. 5, 1998, 60/096,319, filed Aug. 12, 1998, 60/095,454, filed Aug. 6, 1998, and 60/095,455, filed Aug. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/054,988, filed Jan. 25, 2002, which is a continuation of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/097,917, filed Aug. 25, 1998, and 60/098,634, filed Aug. 31, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,893, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/531,119, filed Mar. 20, 2000, which is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/101,546, filed Sep. 23, 1998, and 60/102,895, filed Oct. 2, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,820, filed Sep. 10, 2001, which is a continuation of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999, which claims the benefit of U.S. Provisional Application No. 60/108,207, filed Nov. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/895,298, filed Jul. 2, 2001, which is a continuation of U.S. application Ser. No. 09/591,316, filed Jun. 9, 2000, which is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/113,006, filed Dec. 18, 1998, and 60/112,809, filed Dec. 17, 1998; U.S. Application No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/985,153, filed Nov. 1, 2001, which is a continuation of U.S. application Ser. No. 09/618,150, filed Jul. 17, 2000, which is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000, which claims the benefit of U.S. Provisional Application No. 60/116,330, filed Jan. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/997,131, filed Nov. 30, 2001, which is a continuation of U.S. application Ser. No. 09/628,508, filed Jul. 28, 2000, which is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000, which claims the benefit of U.S. Provisional Application No. 60/119,468, filed Feb. 10, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,882, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/125,055, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,704, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/128,693, filed Apr. 9, 1999, and 60/130,991, filed Apr. 26, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/042,141, filed Jan. 11, 2002, which is a continuation of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000, which claims the benefit of U.S. Provisional Application No. 60/137,725, filed Jun. 7, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/756,168, filed Jan. 9, 2001, which is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 20, 2000, which claims the benefit of U.S. Provisional Application No. 60/145,220, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/060,255, filed Feb. 1, 2002, which is a continuation of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/149,182, filed Aug. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/789,561, filed Feb. 22, 2001, which is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/152,315, filed Sep. 3, 1999, and 60/152,317, filed Sep. 3, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/800,729, filed Mar. 8, 2001, which is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,709, filed Sep. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/832,129, filed Apr. 11, 2001, which is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/163,085, filed Nov. 2, 1999, and 60/172,411, filed Dec. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29363, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,139, filed Jun. 30, 2000, and 60/162,239, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29360, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,138, filed Jun. 30, 2000, and 60/162,211, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29362, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,131, filed Jun. 30, 2000, and 60/162,240, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29365, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,666, filed Jul. 21, 2000, and 60/162,237, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29364, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,134, filed Jun. 30, 2000, and 60/162,238, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30040, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,130, filed Jun. 30, 2000, and 60/163,580, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30037, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,137, filed Jun. 30, 2000, and 60/163,577, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30045, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,133, filed Jun. 30, 2000, and 60/163,581, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30036, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,366, filed Jul. 27, 2000, and 60/163,576, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30039, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,367, filed Jul. 27, 2000, 60/195,296, filed Apr. 7, 2000, and 60/164,344, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30654, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,142, filed Jul. 27, 2000, and 60/164,835, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30628, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,140, filed Jun. 30, 2000, and 60/164,744, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30653, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,193, filed Jul. 27, 2000, and 60/164,735, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30629, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/222,904, filed Aug. 3, 2000, and 60/164,825, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30679, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/224,007, filed Aug. 4, 2000, and 60/164,834, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30674, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,128, filed Jun. 30, 2000, and 60/164,750, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31162, filed Nov. 15, 2000; International Application No. PCT/US00/31162, filed Nov. 15, 2000, claims the benefit of U.S. Provisional Application Nos. 60/215,136, filed Jun. 30, 2000, and 60/166,415, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31282, filed Nov. 15, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,665, filed Jul. 21, 2000, and 60/166,414, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30657, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,132, filed Jun. 30, 2000, and 60/164,731, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01396, filed Jan. 17, 2001; International Application No. PCT/US01/01396, filed Jan. 17, 2001 claims the benefit of U.S. Provisional Application Nos. 60/256,968, filed Dec. 21, 2000, and 60/226,280, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01387, filed Jan. 17, 2001; International Application No. PCT/US01/01387, filed Jan. 17, 2001 claims the benefit of U.S. Provisional Application Nos. 60/259,803, filed Jan. 5, 2001, and 60/226,380, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01567, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/228,084, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01431, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,968, filed Sep. 12, 2000; International Application No. PCT/US01/01431 is a continuation-in-part of U.S. application Ser. No. 09/915,582, filed Jul. 27, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01432, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/236,326, filed Sep. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00544, filed Jan. 9, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,211, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01435, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,282, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01386, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,104, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01565, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,210, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01394, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,805, filed Jan. 5, 2001, and 60/226,278, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01434, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,678, filed Jan. 5, 2001, and 60/226,279, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01397, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,281, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01385, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,969, filed Sep. 12, 2000; U.S. Application No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01384, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,516, filed Jan. 4, 2001, and 60/228,086, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01383, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,804, filed Jan. 5, 2001, and 60/228,083, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05064, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,444, filed Jul. 12, 2001, and 60/270,658, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05301, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,417, filed Jul. 12, 2001, and 60/270,625, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application Nos. 60/304,121, filed Jul. 11, 2001, 60/295,869, filed Jun. 6, 2001, 60/325,209, filed Sep. 28, 2001, 60/311,085, filed Aug. 10, 2001, 60/330,629, filed Oct. 26, 2001, 60/331,046, filed Nov. 7, 2001, 60/358,554, filed Feb. 22, 2002, and 60/358,714, filed Feb. 25, 2002. This application is a continuation-in-part of U.S. application Ser. No. 12/198,817, filed Aug. 26, 2008, which is a divisional of U.S. application Ser. No. 11/346,470, filed Feb. 3, 2006, which is a continuation-in-part of U.S. application Ser. No. 10/472,532, filed Sep. 20, 2003 and accorded a National Stage filing date of Jun. 23, 2004, now abandoned, which is the 35 U.S.C. §371 National Stage of PCT/US02/08278, filed Mar. 19, 2002, which in turn claims benefit of U.S. patent application Ser. No. 10/100,683 (now U.S. Pat. No. 7,368,531, issued May 6, 2008). U.S. application Ser. No. 11/346,470 is also a continuation-in-part of U.S. patent application Ser. No. 10/100,683, filed Mar. 19, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/277,340, filed Mar. 21, 2001, 60/306,171, filed Jul. 19, 2001, and 60/331,287, filed Nov. 13, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/981,876, filed Oct. 19, 2001, which is a divisional of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,161, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,615, filed May 23, 1997, 60/047,600, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/047,595, filed May 23, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, and 60/056,884, filed Aug. 22, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/882,171, filed Jun. 18, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/882,171 is a continuation of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/809,391 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,161, filed Mar. 7, 1997, 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,600, filed May 23, 1997, 60/047,615, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,595, filed May 23, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, 60/056,884, filed Aug. 22, 1997, 60/057,669, filed Sep. 5, 1997, 60/049,610, filed Jun. 13, 1997, 60/061,060, filed Oct. 2, 1997, 60/051,926, filed Jul. 8, 1997, 60/052,874, filed Jul. 16, 1997, 60/058,785, filed Sep. 12, 1997, and 60/055,724, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/058,993, filed Jan. 30, 2002, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. Application No. 09/853,161, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,659 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/853,161 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,797 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,762, filed Mar. 14, 1997, 60/040,710, filed Mar. 14, 1997, 60/050,934, filed May 30, 1997, 60/048,100, filed May 30, 1997, 60/048,357, filed May 30, 1997, 60/048,189, filed May 30, 1997, 60/057,765, filed Sep. 5, 1997, 60/048,970, filed Jun. 6, 1997, and 60/068,368, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/059,395, filed Jan. 31, 2002, which is a divisional of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,245, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,966, filed Oct. 26, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/041,277, filed Mar. 21, 1997, 60/042,344, filed Mar. 21, 1997, 60/041,276, filed Mar. 21, 1997, 60/041,281, filed Mar. 21, 1997, 60/048,094, filed May 30, 1997, 60/048,350, filed May 30, 1997, 60/048,188, filed May 30, 1997, 60/048,135, filed May 30, 1997, 60/050,937, filed May 30, 1997, 60/048,187, filed May 30, 1997, 60/048,099, filed May 30, 1997, 60/048,352, filed May 30, 1997, 60/048,186, filed May 30, 1997, 60/048,069, filed May 30, 1997, 60/048,095, filed May 30, 1997, 60/048,131, filed May 30, 1997, 60/048,096, filed May 30, 1997, 60/048,355, filed May 30, 1997, 60/048,160, filed May 30, 1997, 60/048,351, filed May 30, 1997, 60/048,154, filed May 30, 1997, 60/054,804, filed Aug. 5, 1997, 60/056,370, filed Aug. 19, 1997, and 60/060,862, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/814,122, filed Mar. 22, 2001, which is a continuation of U.S. application Ser. No. 09/577,145, filed May 24, 2000, which is a continuation of U.S. application Ser. No. 09/166,780, filed Oct. 6, 1998, which is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/10868, filed May 28, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/044,039, filed May 30, 1997, 60/048,093, filed May 30, 1997, 60/048,190, filed May 30, 1997, 60/050,935, filed May 30, 1997, 60/048,101, filed May 30, 1997, 60/048,356, filed May 30, 1997, 60/056,250, filed Aug. 29, 1997, 60/056,296, filed Aug. 29, 1997, and 60/056,293, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/11422, filed Jun. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/048,885, filed Jun. 6, 1997, 60/049,375, filed Jun. 6, 1997, 60/048,881, filed Jun. 6, 1997, 60/048,880, filed Jun. 6, 1997, 60/048,896, filed Jun. 6, 1997, 60/049,020, filed Jun. 6, 1997, 60/048,876, filed Jun. 6, 1997, 60/048,895, filed Jun. 6, 1997, 60/048,884, filed Jun. 6, 1997, 60/048,894, filed Jun. 6, 1997, 60/048,971, filed Jun. 6, 1997, 60/048,964, filed Jun. 6, 1997, 60/048,882, filed Jun. 6, 1997, 60/048,899, filed Jun. 6, 1997, 60/048,893, filed Jun. 6, 1997, 60/048,900, filed Jun. 6, 1997, 60/048,901, filed Jun. 6, 1997, 60/048,892, filed Jun. 6, 1997, 60/048,915, filed Jun. 6, 1997, 60/049,019, filed Jun. 6, 1997, 60/048,970, filed Jun. 6, 1997, 60/048,972, filed Jun. 6, 1997, 60/048,916, filed Jun. 6, 1997, 60/049,373, filed Jun. 6, 1997, 60/048,875, filed Jun. 6, 1997, 60/049,374, filed Jun. 6, 1997, 60/048,917, filed Jun. 6, 1997, 60/048,949, filed Jun. 6, 1997, 60/048,974, filed Jun. 6, 1997, 60/048,883, filed Jun. 6, 1997, 60/048,897, filed Jun. 6, 1997, 60/048,898, filed Jun. 6, 1997, 60/048,962, filed Jun. 6, 1997, 60/048,963, filed Jun. 6, 1997, 60/048,877, filed Jun. 6, 1997, 60/048,878, filed Jun. 6, 1997, 60/057,645, filed Sep. 5, 1997, 60/057,642, filed Sep. 5, 1997, 60/057,668, filed Sep. 5, 1997, 60/057,635, filed Sep. 5, 1997, 60/057,627, filed Sep. 5, 1997, 60/057,667, filed Sep. 5, 1997, 60/057,666, filed Sep. 5, 1997, 60/057,764, filed Sep. 5, 1997, 60/057,643, filed Sep. 5, 1997, 60/057,769, filed Sep. 5, 1997, 60/057,763, filed Sep. 5, 1997, 60/057,650, filed Sep. 5, 1997, 60/057,584, filed Sep. 5, 1997, 60/057,647, filed Sep. 5, 1997, 60/057,661, filed Sep. 5, 1997, 60/057,662, filed Sep. 5, 1997, 60/057,646, filed Sep. 5, 1997, 60/057,654, filed Sep. 5, 1997, 60/057,651, filed Sep. 5, 1997, 60/057,644, filed Sep. 5, 1997, 60/057,765, filed Sep. 5, 1997, 60/057,762, filed Sep. 5, 1997, 60/057,775, filed Sep. 5, 1997, 60/057,648, filed Sep. 5, 1997, 60/057,774, filed Sep. 5, 1997, 60/057,649, filed Sep. 5, 1997, 60/057,770, filed Sep. 5, 1997, 60/057,771, filed Sep. 5, 1997, 60/057,761, filed Sep. 5, 1997, 60/057,760, filed Sep. 5, 1997, 60/057,776, filed Sep. 5, 1997, 60/057,778, filed Sep. 5, 1997, 60/057,629, filed Sep. 5, 1997, 60/057,628, filed Sep. 5, 1997, 60/057,777, filed Sep. 5, 1997, 60/057,634, filed Sep. 5, 1997, and 60/070,923, filed Dec. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/05614, filed Feb. 21, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/184,836, filed Feb. 24, 2000, and 60/193,170, filed Mar. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/12125, filed Jun. 11, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/049,547, filed Jun. 13, 1997, 60/049,548, filed Jun. 13, 1997, 60/049,549, filed Jun. 13, 1997, 60/049,550, filed Jun. 13, 1997, 60/049,566, filed Jun. 13, 1997, 60/049,606, filed Jun. 13, 1997, 60/049,607, filed Jun. 13, 1997, 60/049,608, filed Jun. 13, 1997, 60/049,609, filed Jun. 13, 1997, 60/049,610, filed Jun. 13, 1997, 60/049,611, filed Jun. 13, 1997, 60/050,901, filed Jun. 13, 1997, 60/052,989, filed Jun. 13, 1997, 60/051,919, filed Jul. 8, 1997, 60/055,984, filed Aug. 18, 1997, 60/058,665, filed Sep. 12, 1997, 60/058,668, filed Sep. 12, 1997, 60/058,669, filed Sep. 12, 1997, 60/058,750, filed Sep. 12, 1997, 60/058,971, filed Sep. 12, 1997, 60/058,972, filed Sep. 12, 1997, 60/058,975, filed Sep. 12, 1997, 60/060,834, filed Oct. 2, 1997, 60/060,841, filed Oct. 2, 1997, 60/060,844, filed Oct. 2, 1997, 60/060,865, filed Oct. 2, 1997, 60/061,059, filed Oct. 2, 1997, and 60/061,060, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/627,081, filed Jul. 27, 2000, which is a continuation of U.S. application Ser. No. 09/213,365, filed Dec. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,480, filed Jul. 1, 1997, 60/051,381, filed Jul. 1, 1997, 60/058,663, filed Sep. 12, 1997, and 60/058,598, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,490, filed Oct. 30, 2001, which is a divisional of U.S. Application No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,802, filed Oct. 25, 2001, which is a continuation of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/973,278, filed Oct. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,899, filed Oct. 13, 2000; U.S. application Ser. No. 09/973,278 is a continuation-in-part of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,926, filed Jul. 8, 1997, 60/052,793, filed Jul. 8, 1997, 60/051,925, filed Jul. 8, 1997, 60/051,929, filed Jul. 8, 1997, 60/052,803, filed Jul. 8, 1997, 60/052,732, filed Jul. 8, 1997, 60/051,931, filed Jul. 8, 1997, 60/051,932, filed Jul. 8, 1997, 60/051,916, filed Jul. 8, 1997, 60/051,930, filed Jul. 8, 1997, 60/051,918, filed Jul. 8, 1997, 60/051,920, filed Jul. 8, 1997, 60/052,733, filed Jul. 8, 1997, 60/052,795, filed Jul. 8, 1997, 60/051,919, filed Jul. 8, 1997, 60/051,928, filed Jul. 8, 1997, 60/055,722, filed Aug. 18, 1997, 60/055,723, filed Aug. 18, 1997, 60/055,948, filed Aug. 18, 1997, 60/055,949, filed Aug. 18, 1997, 60/055,953, filed Aug. 18, 1997, 60/055,950, filed Aug. 18, 1997, 60/055,947, filed Aug. 18, 1997, 60/055,964, filed Aug. 18, 1997, 60/056,360, filed Aug. 18, 1997, 60/055,684, filed Aug. 18, 1997, 60/055,984, filed Aug. 18, 1997, 60/055,954, filed Aug. 18, 1997, 60/058,785, filed Sep. 12, 1997, 60/058,664, filed Sep. 12, 1997, 60/058,660, filed Sep. 12, 1997, and 60/058,661, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/776,724, filed Feb. 6, 2001, which claims the benefit of U.S. Provisional Application No. 60/180,909, filed Feb. 8, 2000; U.S. application Ser. No. 09/776,724 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/052,661, filed Jul. 16, 1997, 60/052,872, filed Jul. 16, 1997, 60/052,871, filed Jul. 16, 1997, 60/052,874, filed Jul. 16, 1997, 60/052,873, filed Jul. 16, 1997, 60/052,870, filed Jul. 16, 1997, 60/052,875, filed Jul. 16, 1997, 60/053,440, filed Jul. 22, 1997, 60/053,441, filed Jul. 22, 1997, 60/053,442, filed Jul. 22, 1997, 60/056,359, filed Aug. 18, 1997, 60/055,725, filed Aug. 18, 1997, 60/055,985, filed Aug. 18, 1997, 60/055,952, filed Aug. 18, 1997, 60/055,989, filed Aug. 18, 1997, 60/056,361, filed Aug. 18, 1997, 60/055,726, filed Aug. 18, 1997, 60/055,724, filed Aug. 18, 1997, 60/055,946, filed Aug. 18, 1997, and 60/055,683, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/295,558, filed Jun. 5, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,649, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/666,984, filed Sep. 21, 2000, which is a continuation of U.S. application Ser. No. 09/236,557, filed Jan. 26, 1999, which is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/054,212, filed Jul. 30, 1997, 60/054,209, filed Jul. 30, 1997, 60/054,234, filed Jul. 30, 1997, 60/054,218, filed Jul. 30, 1997, 60/054,214, filed Jul. 30, 1997, 60/054,236, filed Jul. 30, 1997, 60/054,215, filed Jul. 30, 1997, 60/054,211, filed Jul. 30, 1997, 60/054,217, filed Jul. 30, 1997, 60/054,213, filed Jul. 30, 1997, 60/055,968, filed Aug. 18, 1997, 60/055,969, filed Aug. 18, 1997, 60/055,972, filed Aug. 18, 1997, 60/056,561, filed Aug. 19, 1997, 60/056,534, filed Aug. 19, 1997, 60/056,729, filed Aug. 19, 1997, 60/056,543, filed Aug. 19, 1997, 60/056,727, filed Aug. 19, 1997, 60/056,554, filed Aug. 19, 1997, and 60/056,730, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which claims the benefit of U.S. Provisional Application No. 60/238,291, filed Oct. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/055,386, filed Aug. 5, 1997, 60/054,807, filed Aug. 5, 1997, 60/055,312, filed Aug. 5, 1997, 60/055,309, filed Aug. 5, 1997, 60/054,798, filed Aug. 5, 1997, 60/055,310, filed Aug. 5, 1997, 60/054,806, filed Aug. 5, 1997, 60/054,809, filed Aug. 5, 1997, 60/054,804, filed Aug. 5, 1997, 60/054,803, filed Aug. 5, 1997, 60/054,808, filed Aug. 5, 1997, 60/055,311, filed Aug. 5, 1997, 60/055,986, filed Aug. 18, 1997, 60/055,970, filed Aug. 18, 1997, 60/056,563, filed Aug. 19, 1997, 60/056,557, filed Aug. 19, 1997, 60/056,731, filed Aug. 19, 1997, 60/056,365, filed Aug. 19, 1997, 60/056,367, filed Aug. 19, 1997, 60/056,370, filed Aug. 19, 1997, 60/056,364, filed Aug. 19, 1997, 60/056,366, filed Aug. 19, 1997, 60/056,732, filed Aug. 19, 1997, and 60/056,371, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,128, filed Nov. 17, 2000, which is a continuation of U.S. application Ser. No. 09/251,329, filed Feb. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,555, filed Aug. 19, 1997, 60/056,556, filed Aug. 19, 1997, 60/056,535, filed Aug. 19, 1997, 60/056,629, filed Aug. 19, 1997, 60/056,369, filed Aug. 19, 1997, 60/056,628, filed Aug. 19, 1997, 60/056,728, filed Aug. 19, 1997, 60/056,368, filed Aug. 19, 1997, 60/056,726, filed Aug. 19, 1997, 60/089,510, filed Jun. 16, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/729,835, filed Dec. 6, 2000, which is a divisional of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,270, filed Aug. 29, 1997, 60/056,271, filed Aug. 29, 1997, 60/056,247, filed Aug. 29, 1997, and 60/056,073, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/047,021, filed Jan. 17, 2002, which is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/01109, filed Jan. 17, 2002, which claims the benefit of U.S. Provisional Application No. 60/262,066, filed Jan. 18, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/057,626, filed Sep. 5, 1997, 60/057,663, filed Sep. 5, 1997, 60/057,669, filed Sep. 5, 1997, 60/058,667, filed Sep. 12, 1997, 60/058,974, filed Sep. 12, 1997, 60/058,973, filed Sep. 12, 1997, 60/058,666, filed Sep. 12, 1997, and 60/090,112, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/281,976, filed Mar. 31, 1999, which is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/060,837, filed Oct. 2, 1997, 60/060,862, filed Oct. 2, 1997, 60/060,839, filed Oct. 2, 1997, 60/060,866, filed Oct. 2, 1997, 60/060,843, filed Oct. 2, 1997, 60/060,836, filed Oct. 2, 1997, 60/060,838, filed Oct. 2, 1997, 60/060,874, filed Oct. 2, 1997, 60/060,833, filed Oct. 2, 1997, 60/060,884, filed Oct. 2, 1997, and 60/060,880, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,429, filed Oct. 30, 2001, which claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 09/984,429 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/061,463, filed Oct. 9, 1997, 60/061,529, filed Oct. 9, 1997, 60/071,498, filed Oct. 9, 1997, 60/061,527, filed Oct. 9, 1997, 60/061,536, filed Oct. 9, 1997, and 60/061,532, filed Oct. 9, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/296,622, filed Apr. 23, 1999, which is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/063,099, filed Oct. 24, 1997, 60/063,088, filed Oct. 24, 1997, 60/063,100, filed Oct. 24, 1997, 60/063,387, filed Oct. 24, 1997, 60/063,148, filed Oct. 24, 1997, 60/063,386, filed Oct. 24, 1997, 60/062,784, filed Oct. 24, 1997, 60/063,091, filed Oct. 24, 1997, 60/063,090, filed Oct. 24, 1997, 60/063,089, filed Oct. 24, 1997, 60/063,092, filed Oct. 24, 1997, 60/063,111, filed Oct. 24, 1997, 60/063,101, filed Oct. 24, 1997, 60/063,109, filed Oct. 24, 1997, 60/063,110, filed Oct. 24, 1997, 60/063,098, filed Oct. 24, 1997, and 60/063,097, filed Oct. 24, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/974,879, filed Oct. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,893, filed Oct. 13, 2000; U.S. application Ser. No. 09/974,879 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/064,911, filed Nov. 7, 1997, 60/064,912, filed Nov. 7, 1997, 60/064,983, filed Nov. 7, 1997, 60/064,900, filed Nov. 7, 1997, 60/064,988, filed Nov. 7, 1997, 60/064,987, filed Nov. 7, 1997, 60/064,908, filed Nov. 7, 1997, 60/064,984, filed Nov. 7, 1997, 60/064,985, filed Nov. 7, 1997, 60/066,094, filed Nov. 17, 1997, 60/066,100, filed Nov. 17, 1997, 60/066,089, filed Nov. 17, 1997, 60/066,095, filed Nov. 17, 1997, and 60/066,090, filed Nov. 17, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/334,595, filed Jun. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/070,923, filed Dec. 18, 1997, 60/068,007, filed Dec. 18, 1997, 60/068,057, filed Dec. 18, 1997, 60/068,006, filed Dec. 18, 1997, 60/068,369, filed Dec. 19, 1997, 60/068,367, filed Dec. 19, 1997, 60/068,368, filed Dec. 19, 1997, 60/068,169, filed Dec. 19, 1997, 60/068,053, filed Dec. 18, 1997, 60/068,064, filed Dec. 18, 1997, 60/068,054, filed Dec. 18, 1997, 60/068,008, filed Dec. 18, 1997, and 60/068,365, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/938,671, filed Aug. 27, 2001, which is a continuation of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/070,704, filed Jan. 7, 1998, 60/070,658, filed Jan. 7, 1998, 60/070,692, filed Jan. 7, 1998, and 60/070,657, filed Jan. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/949,925, filed Sep. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,150, filed Sep. 12, 2000; U.S. application Ser. No. 09/949,925 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 09/949,925 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/813,153, filed Mar. 21, 2001, which is a continuation of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/073,170, filed Jan. 30, 1998, 60/073,167, filed Jan. 30, 1998, 60/073,165, filed Jan. 30, 1998, 60/073,164, filed Jan. 30, 1998, 60/073,162, filed Jan. 30, 1998, 60/073,161, filed Jan. 30, 1998, 60/073,160, filed Jan. 30, 1998, and 60/073,159, filed Jan. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/062,548, filed Feb. 5, 2002, which is a continuation of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/074,118, filed Feb. 9, 1998, 60/074,157, filed Feb. 9, 1998, 60/074,037, filed Feb. 9, 1998, 60/074,141, filed Feb. 9, 1998, and 60/074,341, filed Feb. 9, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,129, filed Nov. 17, 2000, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 09/716,129 is a continuation of U.S. application Ser. No. 09/382,572, filed Aug. 25, 1999, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/076,053, filed Feb. 26, 1998, 60/076,051, filed Feb. 26, 1998, 60/076,054, filed Feb. 26, 1998, 60/076,052, filed Feb. 26, 1998, and 60/076,057, filed Feb. 26, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/798,889, filed Mar. 6, 2001, which is a continuation of U.S. application Ser. No. 09/393,022, filed Sep. 9, 1999, which is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/077,714, filed Mar. 12, 1998, 60/077,686, filed Mar. 12, 1998, 60/077,687, filed Mar. 12, 1998, and 60/077,696, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/397,945, filed Sep. 17, 1999, which is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/078,566, filed Mar. 19, 1998, 60/078,576, filed Mar. 19, 1998, 60/078,573, filed Mar. 19, 1998, 60/078,574, filed Mar. 19, 1998, 60/078,579, filed Mar. 19, 1998, 60/080,314, filed Apr. 1, 1998, 60/080,312, filed Apr. 1, 1998, 60/078,578, filed Mar. 19, 1998, 60/078,581, filed Mar. 19, 1998, 60/078,577, filed Mar. 19, 1998, 60/078,563, filed Mar. 19, 1998, and 60/080,313, filed Apr. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,783, filed Sep. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,846, filed Sep. 11, 2000; U.S. application Ser. No. 09/948,783 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/085,093, filed May 12, 1998, 60/085,094, filed May 12, 1998, 60/085,105, filed May 12, 1998, 60/085,180, filed May 12, 1998, 60/085,927, filed May 18, 1998, 60/085,906, filed May 18, 1998, 60/085,920, filed May 18, 1998, 60/085,924, filed May 18, 1998, 60/085,922, filed May 18, 1998, 60/085,923, filed May 18, 1998, 60/085,921, filed May 18, 1998, 60/085,925, filed May 18, 1998, and 60/085,928, filed May 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,873, filed Jan. 18, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/263,681, filed Jan. 24, 2001, and 60/263,230, filed Jan. 23, 2001; U.S. application Ser. No. 10/050,873 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/012,542, filed Dec. 12, 2001, which is a divisional of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/089,507, filed Jun. 16, 1998, 60/089,508, filed Jun. 16, 1998, 60/089,509, filed Jun. 16, 1998, 60/089,510, filed Jun. 16, 1998, 60/090,112, filed Jun. 22, 1998, and 60/090,113, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,271, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,276, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/092,921, filed Jul. 15, 1998, 60/092,922, filed Jul. 15, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/29871, filed Sep. 24, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,925, filed Sep. 25, 2000; International Application No. PCT/US01/29871 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/350,898, filed Jan. 25, 2002; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/489,847, filed Jan. 24, 2000, which is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/094,657, filed Jul. 30, 1998, 60/095,486, filed Aug. 5, 1998, 60/096,319, filed Aug. 12, 1998, 60/095,454, filed Aug. 6, 1998, and 60/095,455, filed Aug. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/054,988, filed Jan. 25, 2002, which is a continuation of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/097,917, filed Aug. 25, 1998, and 60/098,634, filed Aug. 31, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,893, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/531,119, filed Mar. 20, 2000, which is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/101,546, filed Sep. 23, 1998, and 60/102,895, filed Oct. 2, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,820, filed Sep. 10, 2001, which is a continuation of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999, which claims the benefit of U.S. Provisional Application No. 60/108,207, filed Nov. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/895,298, filed Jul. 2, 2001, which is a continuation of U.S. application Ser. No. 09/591,316, filed Jun. 9, 2000, which is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/113,006, filed Dec. 18, 1998, and 60/112,809, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/985,153, filed Nov. 1, 2001, which is a continuation of U.S. application Ser. No. 09/618,150, filed Jul. 17, 2000, which is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000, which claims the benefit of U.S. Provisional Application No. 60/116,330, filed Jan. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/997,131, filed Nov. 30, 2001, which is a continuation of U.S. application Ser. No. 09/628,508, filed Jul. 28, 2000, which is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000, which claims the benefit of U.S. Provisional Application No. 60/119,468, filed Feb. 10, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,882, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/125,055, filed Mar. 18, 1999; U.S. Application No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,704, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/128,693, filed Apr. 9, 1999, and 60/130,991, filed Apr. 26, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/042,141, filed Jan. 11, 2002, which is a continuation of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000, which claims the benefit of U.S. Provisional Application No. 60/137,725, filed Jun. 7, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/756,168, filed Jan. 9, 2001, which is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 20, 2000, which claims the benefit of U.S. Provisional Application No. 60/145,220, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/060,255, filed Feb. 1, 2002, which is a continuation of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/149,182, filed Aug. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/789,561, filed Feb. 22, 2001, which is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/152,315, filed Sep. 3, 1999, and 60/152,317, filed Sep. 3, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/800,729, filed Mar. 8, 2001, which is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,709, filed Sep. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/832,129, filed Apr. 11, 2001, which is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/163,085, filed Nov. 2, 1999, and 60/172,411, filed Dec. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29363, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,139, filed Jun. 30, 2000, and 60/162,239, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29360, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,138, filed Jun. 30, 2000, and 60/162,211, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29362, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,131, filed Jun. 30, 2000, and 60/162,240, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29365, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,666, filed Jul. 21, 2000, and 60/162,237, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29364, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,134, filed Jun. 30, 2000, and 60/162,238, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30040, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,130, filed Jun. 30, 2000, and 60/163,580, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30037, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,137, filed Jun. 30, 2000, and 60/163,577, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30045, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,133, filed Jun. 30, 2000, and 60/163,581, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30036, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,366, filed Jul. 27, 2000, and 60/163,576, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30039, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,367, filed Jul. 27, 2000, 60/195,296, filed Apr. 7, 2000, and 60/164,344, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30654, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,142, filed Jul. 27, 2000, and 60/164,835, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30628, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,140, filed Jun. 30, 2000, and 60/164,744, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30653, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,193, filed Jul. 27, 2000, and 60/164,735, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30629, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/222,904, filed Aug. 3, 2000, and 60/164,825, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30679, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/224,007, filed Aug. 4, 2000, and 60/164,834, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30674, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,128, filed Jun. 30, 2000, and 60/164,750, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31162, filed Nov. 15, 2000; International Application No. PCT/US00/31162, filed Nov. 15, 2000 claims the benefit of U.S. Provisional Application Nos. 60/215,136, and 60/166,415, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31282, filed Nov. 15, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,665, filed Jul. 21, 2000, and 60/166,414, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30657, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,132, filed Jun. 30, 2000, and 60/164,731, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01396, filed Jan. 17, 2001; International Application No. PCT/US01/01396, filed Jan. 17, 2001 claims the benefit of U.S. Provisional Application Nos. 60/256,968, filed Dec. 21, 2000, and 60/226,280, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01387, filed Jan. 17, 2001; International Application No. PCT/US01/01387, filed Jan. 17, 2001 claims the benefit of U.S. Provisional Application Nos. 60/259,803, filed Jan. 5, 2001, and 60/226,380, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01567, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/228,084, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01431, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,968, filed Sep. 12, 2000; International Application No. PCT/US01/01431 is a continuation-in-part of U.S. application Ser. No. 09/915,582, filed Jul. 27, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01432, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/236,326, filed Sep. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00544, filed Jan. 9, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,211, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01435, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,282, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01386, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,104, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01565, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,210, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01394, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,805, filed Jan. 5, 2001, and 60/226,278, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01434, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,678, filed Jan. 5, 2001, and 60/226,279, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01397, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,281, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01385, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,969, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01384, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,516, filed Jan. 4, 2001, and 60/228,086, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01383, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,804, filed Jan. 5, 2001, and 60/228,083, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05064, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,444, filed Jul. 12, 2001, and 60/270,658, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05301, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,417, filed Jul. 12, 2001, and 60/270,625, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application Nos. 60/304,121, filed Jul. 11, 2001, 60/295,869, filed Jun. 6, 2001, 60/325,209, filed Sep. 28, 2001, 60/311,085, filed Aug. 10, 2001, 60/330,629, filed Oct. 26, 2001, 60/331,046, filed Nov. 7, 2001, 60/358,554, filed Feb. 22, 2002, and 60/358,714, filed Feb. 25, 2002. This application is a continuation-in-part of U.S. application Ser. No. 11/689,173, filed Mar. 21, 2007, which is a continuation of U.S. application Ser. No. 11/001,793, filed Dec. 2, 2004, which is a divisional of U.S. application Ser. No. 10/100,683, filed Mar. 19, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/277,340, filed Mar. 21, 2001, 60/306,171, filed Jul. 19, 2001, and 60/331,287, filed Nov. 13, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/981,876, filed Oct. 19, 2001, which is a divisional of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/621,011, filed Jul. 20, 2000, which is a continuation of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/148,545, filed Sep. 4, 1998, which is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04482, filed Mar. 6, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,161, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,615, filed May 23, 1997, 60/047,600, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/047,595, filed May 23, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, and 60/056,884, filed Aug. 22, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/882,171, filed Jun. 18, 2001, which is a continuation of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, which claims the benefit of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 09/809,391 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, which is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04493, filed Mar. 6, 1998; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/040,161, filed Mar. 7, 1997; International Application No. PCT/US98/04493 claims the benefit of U.S. Provisional Application Nos. 60/040,162, filed Mar. 7, 1997, 60/040,333, filed Mar. 7, 1997, 60/038,621, filed Mar. 7, 1997, 60/040,626, filed Mar. 7, 1997, 60/040,334, filed Mar. 7, 1997, 60/040,336, filed Mar. 7, 1997, 60/040,163, filed Mar. 7, 1997, 60/047,600, filed May 23, 1997, 60/047,615, filed May 23, 1997, 60/047,597, filed May 23, 1997, 60/047,502, filed May 23, 1997, 60/047,633, filed May 23, 1997, 60/047,583, filed May 23, 1997, 60/047,617, filed May 23, 1997, 60/047,618, filed May 23, 1997, 60/047,503, filed May 23, 1997, 60/047,592, filed May 23, 1997, 60/047,581, filed May 23, 1997, 60/047,584, filed May 23, 1997, 60/047,500, filed May 23, 1997, 60/047,587, filed May 23, 1997, 60/047,492, filed May 23, 1997, 60/047,598, filed May 23, 1997, 60/047,613, filed May 23, 1997, 60/047,582, filed May 23, 1997, 60/047,596, filed May 23, 1997, 60/047,612, filed May 23, 1997, 60/047,632, filed May 23, 1997, 60/047,601, filed May 23, 1997, 60/043,580, filed Apr. 11, 1997, 60/043,568, filed Apr. 11, 1997, 60/043,314, filed Apr. 11, 1997, 60/043,569, filed Apr. 11, 1997, 60/043,311, filed Apr. 11, 1997, 60/043,671, filed Apr. 11, 1997, 60/043,674, filed Apr. 11, 1997, 60/043,669, filed Apr. 11, 1997, 60/043,312, filed Apr. 11, 1997, 60/043,313, filed Apr. 11, 1997, 60/043,672, filed Apr. 11, 1997, 60/043,315, filed Apr. 11, 1997, 60/048,974, filed Jun. 6, 1997, 60/056,886, filed Aug. 22, 1997, 60/056,877, filed Aug. 22, 1997, 60/056,889, filed Aug. 22, 1997, 60/056,893, filed Aug. 22, 1997, 60/056,630, filed Aug. 22, 1997, 60/056,878, filed Aug. 22, 1997, 60/056,662, filed Aug. 22, 1997, 60/056,872, filed Aug. 22, 1997, 60/056,882, filed Aug. 22, 1997, 60/056,637, filed Aug. 22, 1997, 60/056,903, filed Aug. 22, 1997, 60/056,888, filed Aug. 22, 1997, 60/056,879, filed Aug. 22, 1997, 60/056,880, filed Aug. 22, 1997, 60/056,894, filed Aug. 22, 1997, 60/056,911, filed Aug. 22, 1997, 60/056,636, filed Aug. 22, 1997, 60/056,874, filed Aug. 22, 1997, 60/056,910, filed Aug. 22, 1997, 60/056,864, filed Aug. 22, 1997, 60/056,631, filed Aug. 22, 1997, 60/056,845, filed Aug. 22, 1997, 60/056,892, filed Aug. 22, 1997, 60/057,761, filed Sep. 5, 1997, 60/047,595, filed May 23, 1997, 60/047,599, filed May 23, 1997, 60/047,588, filed May 23, 1997, 60/047,585, filed May 23, 1997, 60/047,586, filed May 23, 1997, 60/047,590, filed May 23, 1997, 60/047,594, filed May 23, 1997, 60/047,589, filed May 23, 1997, 60/047,593, filed May 23, 1997, 60/047,614, filed May 23, 1997, 60/043,578, filed Apr. 11, 1997, 60/043,576, filed Apr. 11, 1997, 60/047,501, filed May 23, 1997, 60/043,670, filed Apr. 11, 1997, 60/056,632, filed Aug. 22, 1997, 60/056,664, filed Aug. 22, 1997, 60/056,876, filed Aug. 22, 1997, 60/056,881, filed Aug. 22, 1997, 60/056,909, filed Aug. 22, 1997, 60/056,875, filed Aug. 22, 1997, 60/056,862, filed Aug. 22, 1997, 60/056,887, filed Aug. 22, 1997, 60/056,908, filed Aug. 22, 1997, 60/048,964, filed Jun. 6, 1997, 60/057,650, filed Sep. 5, 1997, 60/056,884, filed Aug. 22, 1997, 60/057,669, filed Sep. 5, 1997, 60/049,610, filed Jun. 13, 1997, 60/061,060, filed Oct. 2, 1997, 60/051,926, filed Jul. 8, 1997, 60/052,874, filed Jul. 16, 1997, 60/058,785, filed Sep. 12, 1997, and 60/055,724, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/058,993, filed Jan. 30, 2002, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/058,993 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,659, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,659 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/853,161, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/853,161 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/852,797, filed May 11, 2001, which claims the benefit of U.S. Provisional Application No. 60/265,583, filed Feb. 2, 2001; U.S. application Ser. No. 09/852,797 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/152,060, filed Sep. 11, 1998, which is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/04858, filed Mar. 12, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/040,762, filed Mar. 14, 1997, 60/040,710, filed Mar. 14, 1997, 60/050,934, filed May 30, 1997, 60/048,100, filed May 30, 1997, 60/048,357, filed May 30, 1997, 60/048,189, filed May 30, 1997, 60/057,765, filed Sep. 5, 1997, 60/048,970, filed Jun. 6, 1997, and 60/068,368, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/059,395, filed Jan. 31, 2002, which is a divisional of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,245, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,966, filed Oct. 26, 2001, which is a divisional of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/966,262, filed Oct. 1, 2001, which is a continuation of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/154,707, filed Sep. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/05311, filed Mar. 19, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/041,277, filed Mar. 21, 1997, 60/042,344, filed Mar. 21, 1997, 60/041,276, filed Mar. 21, 1997, 60/041,281, filed Mar. 21, 1997, 60/048,094, filed May 30, 1997, 60/048,350, filed May 30, 1997, 60/048,188, filed May 30, 1997, 60/048,135, filed May 30, 1997, 60/050,937, filed May 30, 1997, 60/048,187, filed May 30, 1997, 60/048,099, filed May 30, 1997, 60/048,352, filed May 30, 1997, 60/048,186, filed May 30, 1997, 60/048,069, filed May 30, 1997, 60/048,095, filed May 30, 1997, 60/048,131, filed May 30, 1997, 60/048,096, filed May 30, 1997, 60/048,355, filed May 30, 1997, 60/048,160, filed May 30, 1997, 60/048,351, filed May 30, 1997, 60/048,154, filed May 30, 1997, 60/054,804, filed Aug. 5, 1997, 60/056,370, filed Aug. 19, 1997, and 60/060,862, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/814,122, filed Mar. 22, 2001, which is a continuation of U.S. application Ser. No. 09/577,145, filed May 24, 2000, which is a continuation of U.S. application Ser. No. 09/166,780, filed Oct. 6, 1998, which is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/06801, filed Apr. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/042,726, filed Apr. 8, 1997, 60/042,727, filed Apr. 8, 1997, 60/042,728, filed Apr. 8, 1997, 60/042,754, filed Apr. 8, 1997, 60/042,825, filed Apr. 8, 1997, 60/048,068, filed May 30, 1997, 60/048,070, filed May 30, 1997, and 60/048,184, filed May 30, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/10868, filed May 28, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/044,039, filed May 30, 1997, 60/048,093, filed May 30, 1997, 60/048,190, filed May 30, 1997, 60/050,935, filed May 30, 1997, 60/048,101, filed May 30, 1997, 60/048,356, filed May 30, 1997, 60/056,250, filed Aug. 29, 1997, 60/056,296, filed Aug. 29, 1997, and 60/056,293, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/11422, filed Jun. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/048,885, filed Jun. 6, 1997, 60/049,375, filed Jun. 6, 1997, 60/048,881, filed Jun. 6, 1997, 60/048,880, filed Jun. 6, 1997, 60/048,896, filed Jun. 6, 1997, 60/049,020, filed Jun. 6, 1997, 60/048,876, filed Jun. 6, 1997, 60/048,895, filed Jun. 6, 1997, 60/048,884, filed Jun. 6, 1997, 60/048,894, filed Jun. 6, 1997, 60/048,971, filed Jun. 6, 1997, 60/048,964, filed Jun. 6, 1997, 60/048,882, filed Jun. 6, 1997, 60/048,899, filed Jun. 6, 1997, 60/048,893, filed Jun. 6, 1997, 60/048,900, filed Jun. 6, 1997, 60/048,901, filed Jun. 6, 1997, 60/048,892, filed Jun. 6, 1997, 60/048,915, filed Jun. 6, 1997, 60/049,019, filed Jun. 6, 1997, 60/048,970, filed Jun. 6, 1997, 60/048,972, filed Jun. 6, 1997, 60/048,916, filed Jun. 6, 1997, 60/049,373, filed Jun. 6, 1997, 60/048,875, filed Jun. 6, 1997, 60/049,374, filed Jun. 6, 1997, 60/048,917, filed Jun. 6, 1997, 60/048,949, filed Jun. 6, 1997, 60/048,974, filed Jun. 6, 1997, 60/048,883, filed Jun. 6, 1997, 60/048,897, filed Jun. 6, 1997, 60/048,898, filed Jun. 6, 1997, 60/048,962, filed Jun. 6, 1997, 60/048,963, filed Jun. 6, 1997, 60/048,877, filed Jun. 6, 1997, 60/048,878, filed Jun. 6, 1997, 60/057,645, filed Sep. 5, 1997, 60/057,642, filed Sep. 5, 1997, 60/057,668, filed Sep. 5, 1997, 60/057,635, filed Sep. 5, 1997, 60/057,627, filed Sep. 5, 1997, 60/057,667, filed Sep. 5, 1997, 60/057,666, filed Sep. 5, 1997, 60/057,764, filed Sep. 5, 1997, 60/057,643, filed Sep. 5, 1997, 60/057,769, filed Sep. 5, 1997, 60/057,763, filed Sep. 5, 1997, 60/057,650, filed Sep. 5, 1997, 60/057,584, filed Sep. 5, 1997, 60/057,647, filed Sep. 5, 1997, 60/057,661, filed Sep. 5, 1997, 60/057,662, filed Sep. 5, 1997, 60/057,646, filed Sep. 5, 1997, 60/057,654, filed Sep. 5, 1997, 60/057,651, filed Sep. 5, 1997, 60/057,644, filed Sep. 5, 1997, 60/057,765, filed Sep. 5, 1997, 60/057,762, filed Sep. 5, 1997, 60/057,775, filed Sep. 5, 1997, 60/057,648, filed Sep. 5, 1997, 60/057,774, filed Sep. 5, 1997, 60/057,649, filed Sep. 5, 1997, 60/057,770, filed Sep. 5, 1997, 60/057,771, filed Sep. 5, 1997, 60/057,761, filed Sep. 5, 1997, 60/057,760, filed Sep. 5, 1997, 60/057,776, filed Sep. 5, 1997, 60/057,778, filed Sep. 5, 1997, 60/057,629, filed Sep. 5, 1997, 60/057,628, filed Sep. 5, 1997, 60/057,777, filed Sep. 5, 1997, 60/057,634, filed Sep. 5, 1997, and 60/070,923, filed Dec. 18, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/05614, filed Feb. 21, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/184,836, filed Feb. 24, 2000, and 60/193,170, filed Mar. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/12125, filed Jun. 11, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/049,547, filed Jun. 13, 1997, 60/049,548, filed Jun. 13, 1997, 60/049,549, filed Jun. 13, 1997, 60/049,550, filed Jun. 13, 1997, 60/049,566, filed Jun. 13, 1997, 60/049,606, filed Jun. 13, 1997, 60/049,607, filed Jun. 13, 1997, 60/049,608, filed Jun. 13, 1997, 60/049,609, filed Jun. 13, 1997, 60/049,610, filed Jun. 13, 1997, 60/049,611, filed Jun. 13, 1997, 60/050,901, filed Jun. 13, 1997, 60/052,989, filed Jun. 13, 1997, 60/051,919, filed Jul. 8, 1997, 60/055,984, filed Aug. 18, 1997, 60/058,665, filed Sep. 12, 1997, 60/058,668, filed Sep. 12, 1997, 60/058,669, filed Sep. 12, 1997, 60/058,750, filed Sep. 12, 1997, 60/058,971, filed Sep. 12, 1997, 60/058,972, filed Sep. 12, 1997, 60/058,975, filed Sep. 12, 1997, 60/060,834, filed Oct. 2, 1997, 60/060,841, filed Oct. 2, 1997, 60/060,844, filed Oct. 2, 1997, 60/060,865, filed Oct. 2, 1997, 60/061,059, filed Oct. 2, 1997, and 60/061,060, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. Application No. 09/627,081, filed Jul. 27, 2000, which is a continuation of U.S. application Ser. No. 09/213,365, filed Dec. 17, 1998, which is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13608, filed Jun. 30, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,480, filed Jul. 1, 1997, 60/051,381, filed Jul. 1, 1997, 60/058,663, filed Sep. 12, 1997, and 60/058,598, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,490, filed Oct. 30, 2001, which is a divisional of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/983,802, filed Oct. 25, 2001, which is a continuation of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/973,278, filed Oct. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,899, filed Oct. 13, 2000; U.S. application Ser. No. 09/973,278 is a continuation-in-part of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/051,926, filed Jul. 8, 1997, 60/052,793, filed Jul. 8, 1997, 60/051,925, filed Jul. 8, 1997, 60/051,929, filed Jul. 8, 1997, 60/052,803, filed Jul. 8, 1997, 60/052,732, filed Jul. 8, 1997, 60/051,931, filed Jul. 8, 1997, 60/051,932, filed Jul. 8, 1997, 60/051,916, filed Jul. 8, 1997, 60/051,930, filed Jul. 8, 1997, 60/051,918, filed Jul. 8, 1997, 60/051,920, filed Jul. 8, 1997, 60/052,733, filed Jul. 8, 1997, 60/052,795, filed Jul. 8, 1997, 60/051,919, filed Jul. 8, 1997, 60/051,928, filed Jul. 8, 1997, 60/055,722, filed Aug. 18, 1997, 60/055,723, filed Aug. 18, 1997, 60/055,948, filed Aug. 18, 1997, 60/055,949, filed Aug. 18, 1997, 60/055,953, filed Aug. 18, 1997, 60/055,950, filed Aug. 18, 1997, 60/055,947, filed Aug. 18, 1997, 60/055,964, filed Aug. 18, 1997, 60/056,360, filed Aug. 18, 1997, 60/055,684, filed Aug. 18, 1997, 60/055,984, filed Aug. 18, 1997, 60/055,954, filed Aug. 18, 1997, 60/058,785, filed Sep. 12, 1997, 60/058,664, filed Sep. 12, 1997, 60/058,660, filed Sep. 12, 1997, and 60/058,661, filed Sep. 12, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. Application No. 09/776,724, filed Feb. 6, 2001, which claims the benefit of U.S. Provisional Application No. 60/180,909, filed Feb. 8, 2000; U.S. application Ser. No. 09/776,724 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/669,688, filed Sep. 26, 2000, which is a continuation of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/229,982, filed Jan. 14, 1999, which is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/14613, filed Jul. 15, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/052,661, filed Jul. 16, 1997, 60/052,872, filed Jul. 16, 1997, 60/052,871, filed Jul. 16, 1997, 60/052,874, filed Jul. 16, 1997, 60/052,873, filed Jul. 16, 1997, 60/052,870, filed Jul. 16, 1997, 60/052,875, filed Jul. 16, 1997, 60/053,440, filed Jul. 22, 1997, 60/053,441, filed Jul. 22, 1997, 60/053,442, filed Jul. 22, 1997, 60/056,359, filed Aug. 18, 1997, 60/055,725, filed Aug. 18, 1997, 60/055,985, filed Aug. 18, 1997, 60/055,952, filed Aug. 18, 1997, 60/055,989, filed Aug. 18, 1997, 60/056,361, filed Aug. 18, 1997, 60/055,726, filed Aug. 18, 1997, 60/055,724, filed Aug. 18, 1997, 60/055,946, filed Aug. 18, 1997, and 60/055,683, filed Aug. 18, 1997; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/295,558, filed Jun. 5, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,649, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/666,984, filed Sep. 21, 2000, which is a continuation of U.S. application Ser. No. 09/236,557, filed Jan. 26, 1999, which is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/15949, filed Jul. 29, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/054,212, filed Jul. 30, 1997, 60/054,209, filed Jul. 30, 1997, 60/054,234, filed Jul. 30, 1997, 60/054,218, filed Jul. 30, 1997, 60/054,214, filed Jul. 30, 1997, 60/054,236, filed Jul. 30, 1997, 60/054,215, filed Jul. 30, 1997, 60/054,211, filed Jul. 30, 1997, 60/054,217, filed Jul. 30, 1997, 60/054,213, filed Jul. 30, 1997, 60/055,968, filed Aug. 18, 1997, 60/055,969, filed Aug. 18, 1997, 60/055,972, filed Aug. 18, 1997, 60/056,561, filed Aug. 19, 1997, 60/056,534, filed Aug. 19, 1997, 60/056,729, filed Aug. 19, 1997, 60/056,543, filed Aug. 19, 1997, 60/056,727, filed Aug. 19, 1997, 60/056,554, filed Aug. 19, 1997, and 60/056,730, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/774,639, filed Feb. 1, 2001, which is a continuation of U.S. application Ser. No. 09/244,112, filed Feb. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/969,730, filed Oct. 4, 2001, which claims the benefit of U.S. Provisional Application No. 60/238,291, filed Oct. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/16235, filed Aug. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/055,386, filed Aug. 5, 1997, 60/054,807, filed Aug. 5, 1997, 60/055,312, filed Aug. 5, 1997, 60/055,309, filed Aug. 5, 1997, 60/054,798, filed Aug. 5, 1997, 60/055,310, filed Aug. 5, 1997, 60/054,806, filed Aug. 5, 1997, 60/054,809, filed Aug. 5, 1997, 60/054,804, filed Aug. 5, 1997, 60/054,803, filed Aug. 5, 1997, 60/054,808, filed Aug. 5, 1997, 60/055,311, filed Aug. 5, 1997, 60/055,986, filed Aug. 18, 1997, 60/055,970, filed Aug. 18, 1997, 60/056,563, filed Aug. 19, 1997, 60/056,557, filed Aug. 19, 1997, 60/056,731, filed Aug. 19, 1997, 60/056,365, filed Aug. 19, 1997, 60/056,367, filed Aug. 19, 1997, 60/056,370, filed Aug. 19, 1997, 60/056,364, filed Aug. 19, 1997, 60/056,366, filed Aug. 19, 1997, 60/056,732, filed Aug. 19, 1997, and 60/056,371, filed Aug. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,128, filed Nov. 17, 2000, which is a continuation of U.S. application Ser. No. 09/251,329, filed Feb. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17044, filed Aug. 18, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,555, filed Aug. 19, 1997, 60/056,556, filed Aug. 19, 1997, 60/056,535, filed Aug. 19, 1997, 60/056,629, filed Aug. 19, 1997, 60/056,369, filed Aug. 19, 1997, 60/056,628, filed Aug. 19, 1997, 60/056,728, filed Aug. 19, 1997, 60/056,368, filed Aug. 19, 1997, 60/056,726, filed Aug. 19, 1997, 60/089,510, filed Jun. 16, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/729,835, filed Dec. 6, 2000, which is a divisional of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/257,179, filed Feb. 25, 1999, which is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/17709, filed Aug. 27, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/056,270, filed Aug. 29, 1997, 60/056,271, filed Aug. 29, 1997, 60/056,247, filed Aug. 29, 1997, and 60/056,073, filed Aug. 29, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/047,021, filed Jan. 17, 2002, which is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/722,329, filed Nov. 28, 2000, which is a continuation of U.S. application Ser. No. 09/262,109, filed Mar. 4, 1999, which is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/01109, filed Jan. 17, 2002, which claims the benefit of U.S. Provisional Application No. 60/262,066, filed Jan. 18, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/18360, filed Sep. 3, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/057,626, filed Sep. 5, 1997, 60/057,663, filed Sep. 5, 1997, 60/057,669, filed Sep. 5, 1997, 60/058,667, filed Sep. 12, 1997, 60/058,974, filed Sep. 12, 1997, 60/058,973, filed Sep. 12, 1997, 60/058,666, filed Sep. 12, 1997, and 60/090,112, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/281,976, filed Mar. 31, 1999, which is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/20775, filed Oct. 1, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/060,837, filed Oct. 2, 1997, 60/060,862, filed Oct. 2, 1997, 60/060,839, filed Oct. 2, 1997, 60/060,866, filed Oct. 2, 1997, 60/060,843, filed Oct. 2, 1997, 60/060,836, filed Oct. 2, 1997, 60/060,838, filed Oct. 2, 1997, 60/060,874, filed Oct. 2, 1997, 60/060,833, filed Oct. 2, 1997, 60/060,884, filed Oct. 2, 1997, and 60/060,880, filed Oct. 2, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,429, filed Oct. 30, 2001, which claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 09/984,429 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/244,591, filed Nov. 1, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/288,143, filed Apr. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/21142, filed Oct. 8, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/061,463, filed Oct. 9, 1997, 60/061,529, filed Oct. 9, 1997, 60/071,498, filed Oct. 9, 1997, 60/061,527, filed Oct. 9, 1997, 60/061,536, filed Oct. 9, 1997, and 60/061,532, filed Oct. 9, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/296,622, filed Apr. 23, 1999, which is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/22376, filed Oct. 23, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/063,099, filed Oct. 24, 1997, 60/063,088, filed Oct. 24, 1997, 60/063,100, filed Oct. 24, 1997, 60/063,387, filed Oct. 24, 1997, 60/063,148, filed Oct. 24, 1997, 60/063,386, filed Oct. 24, 1997, 60/062,784, filed Oct. 24, 1997, 60/063,091, filed Oct. 24, 1997, 60/063,090, filed Oct. 24, 1997, 60/063,089, filed Oct. 24, 1997, 60/063,092, filed Oct. 24, 1997, 60/063,111, filed Oct. 24, 1997, 60/063,101, filed Oct. 24, 1997, 60/063,109, filed Oct. 24, 1997, 60/063,110, filed Oct. 24, 1997, 60/063,098, filed Oct. 24, 1997, and 60/063,097, filed Oct. 24, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/974,879, filed Oct. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/239,893, filed Oct. 13, 2000; U.S. application Ser. No. 09/974,879 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/818,683, filed Mar. 28, 2001, which is a continuation of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/305,736, filed May 5, 1999, which is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/23435, filed Nov. 4, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/064,911, filed Nov. 7, 1997, 60/064,912, filed Nov. 7, 1997, 60/064,983, filed Nov. 7, 1997, 60/064,900, filed Nov. 7, 1997, 60/064,988, filed Nov. 7, 1997, 60/064,987, filed Nov. 7, 1997, 60/064,908, filed Nov. 7, 1997, 60/064,984, filed Nov. 7, 1997, 60/064,985, filed Nov. 7, 1997, 60/066,094, filed Nov. 17, 1997, 60/066,100, filed Nov. 17, 1997, 60/066,089, filed Nov. 17, 1997, 60/066,095, filed Nov. 17, 1997, and 60/066,090, filed Nov. 17, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/334,595, filed Jun. 17, 1999, which is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US98/27059, filed Dec. 17, 1998, which claims the benefit of U.S. Provisional Application Nos. 60/070,923, filed Dec. 18, 1997, 60/068,007, filed Dec. 18, 1997, 60/068,057, filed Dec. 18, 1997, 60/068,006, filed Dec. 18, 1997, 60/068,369, filed Dec. 19, 1997, 60/068,367, filed Dec. 19, 1997, 60/068,368, filed Dec. 19, 1997, 60/068,169, filed Dec. 19, 1997, 60/068,053, filed Dec. 18, 1997, 60/068,064, filed Dec. 18, 1997, 60/068,054, filed Dec. 18, 1997, 60/068,008, filed Dec. 18, 1997, and 60/068,365, filed Dec. 19, 1997; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/938,671, filed Aug. 27, 2001, which is a continuation of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/739,907, filed Dec. 20, 2000, which is a continuation of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/348,457, filed Jul. 7, 1999, which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/070,704, filed Jan. 7, 1998, 60/070,658, filed Jan. 7, 1998, 60/070,692, filed Jan. 7, 1998, and 60/070,657, filed Jan. 7, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/949,925, filed Sep. 12, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,150, filed Sep. 12, 2000; U.S. application Ser. No. 09/949,925 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 09/949,925 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/813,153, filed Mar. 21, 2001, which is a continuation of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/363,044, filed Jul. 29, 1999, which is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/01621, filed Jan. 27, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/073,170, filed Jan. 30, 1998, 60/073,167, filed Jan. 30, 1998, 60/073,165, filed Jan. 30, 1998, 60/073,164, filed Jan. 30, 1998, 60/073,162, filed Jan. 30, 1998, 60/073,161, filed Jan. 30, 1998, 60/073,160, filed Jan. 30, 1998, and 60/073,159, filed Jan. 30, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/062,548, filed Feb. 5, 2002, which is a continuation of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999; U.S. application Ser. No. 09/369,247 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/02293, filed Feb. 4, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/074,118, filed Feb. 9, 1998, 60/074,157, filed Feb. 9, 1998, 60/074,037, filed Feb. 9, 1998, 60/074,141, filed Feb. 9, 1998, and 60/074,341, filed Feb. 9, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/716,129, filed Nov. 17, 2000, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 09/716,129 is a continuation of U.S. application Ser. No. 09/382,572, filed Aug. 25, 1999, which is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/03939, filed Feb. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/076,053, filed Feb. 26, 1998, 60/076,051, filed Feb. 26, 1998, 60/076,054, filed Feb. 26, 1998, 60/076,052, filed Feb. 26, 1998, and 60/076,057, filed Feb. 26, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/798,889, filed Mar. 6, 2001, which is a continuation of U.S. application Ser. No. 09/393,022, filed Sep. 9, 1999, which is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05721, filed Mar. 11, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/077,714, filed Mar. 12, 1998, 60/077,686, filed Mar. 12, 1998, 60/077,687, filed Mar. 12, 1998, and 60/077,696, filed Mar. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/397,945, filed Sep. 17, 1999, which is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/05804, filed Mar. 18, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/078,566, filed Mar. 19, 1998, 60/078,576, filed Mar. 19, 1998, 60/078,573, filed Mar. 19, 1998, 60/078,574, filed Mar. 19, 1998, 60/078,579, filed Mar. 19, 1998, 60/080,314, filed Apr. 1, 1998, 60/080,312, filed Apr. 1, 1998, 60/078,578, filed Mar. 19, 1998, 60/078,581, filed Mar. 19, 1998, 60/078,577, filed Mar. 19, 1998, 60/078,563, filed Mar. 19, 1998, and 60/080,313, filed Apr. 1, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,783, filed Sep. 10, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,846, filed Sep. 11, 2000; U.S. application Ser. No. 09/948,783 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/892,877, filed Jun. 28, 2001, which is a continuation of U.S. application Ser. No. 09/437,658, filed Nov. 10, 1999, which is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/09847, filed May 6, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/085,093, filed May 12, 1998, 60/085,094, filed May 12, 1998, 60/085,105, filed May 12, 1998, 60/085,180, filed May 12, 1998, 60/085,927, filed May 18, 1998, 60/085,906, filed May 18, 1998, 60/085,920, filed May 18, 1998, 60/085,924, filed May 18, 1998, 60/085,922, filed May 18, 1998, 60/085,923, filed May 18, 1998, 60/085,921, filed May 18, 1998, 60/085,925, filed May 18, 1998, and 60/085,928, filed May 18, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,873, filed Jan. 18, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/263,681, filed Jan. 24, 2001, and 60/263,230, filed Jan. 23, 2001; U.S. application Ser. No. 10/050,873 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/012,542, filed Dec. 12, 2001, which is a divisional of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/461,325, filed Dec. 14, 1999, which is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/13418, filed Jun. 15, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/089,507, filed Jun. 16, 1998, 60/089,508, filed Jun. 16, 1998, 60/089,509, filed Jun. 16, 1998, 60/089,510, filed Jun. 16, 1998, 60/090,112, filed Jun. 22, 1998, and 60/090,113, filed Jun. 22, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,271, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/984,276, filed Oct. 29, 2001, which is a divisional of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000, which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/092,921, filed Jul. 15, 1998, 60/092,922, filed Jul. 15, 1998, and 60/092,956, filed Jul. 15, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/29871, filed Sep. 24, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,925, filed Sep. 25, 2000; International Application No. PCT/US01/29871 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001, which is a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application No. 60/350,898, filed Jan. 25, 2002; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/489,847, filed Jan. 24, 2000, which is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/17130, filed Jul. 29, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/094,657, filed Jul. 30, 1998, 60/095,486, filed Aug. 5, 1998, 60/096,319, filed Aug. 12, 1998, 60/095,454, filed Aug. 6, 1998, and 60/095,455, filed Aug. 6, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/054,988, filed Jan. 25, 2002, which is a continuation of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/904,615, filed Jul. 16, 2001, which is a continuation of U.S. application Ser. No. 09/739,254, filed Dec. 19, 2000, which is a continuation of U.S. application Ser. No. 09/511,554, filed Feb. 23, 2000, which is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/19330, filed Aug. 24, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/097,917, filed Aug. 25, 1998, and 60/098,634, filed Aug. 31, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/820,893, filed Mar. 30, 2001, which is a continuation of U.S. application Ser. No. 09/531,119, filed Mar. 20, 2000, which is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/22012, filed Sep. 22, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/101,546, filed Sep. 23, 1998, and 60/102,895, filed Oct. 2, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/948,820, filed Sep. 10, 2001, which is a continuation of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/565,391, filed May 5, 2000, which is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/26409, filed Nov. 9, 1999, which claims the benefit of U.S. Provisional Application No. 60/108,207, filed Nov. 12, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/895,298, filed Jul. 2, 2001, which is a continuation of U.S. application Ser. No. 09/591,316, filed Jun. 9, 2000, which is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US99/29950, filed Dec. 16, 1999, which claims the benefit of U.S. Provisional Application Nos. 60/113,006, filed Dec. 18, 1998, and 60/112,809, filed Dec. 17, 1998; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/985,153, filed Nov. 1, 2001, which is a continuation of U.S. application Ser. No. 09/618,150, filed Jul. 17, 2000, which is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/00903, filed Jan. 18, 2000, which claims the benefit of U.S. Provisional Application No. 60/116,330, filed Jan. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/997,131, filed Nov. 30, 2001, which is a continuation of U.S. application Ser. No. 09/628,508, filed Jul. 28, 2000, which is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/03062, filed Feb. 8, 2000, which claims the benefit of U.S. Provisional Application No. 60/119,468, filed Feb. 10, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,882, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/661,453, filed Sep. 13, 2000, which is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/06783, filed Mar. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/125,055, filed Mar. 18, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/050,704, filed Jan. 18, 2002, which is a continuation of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/684,524, filed Oct. 10, 2000, which is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/08979, filed Apr. 6, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/128,693, filed Apr. 9, 1999, and 60/130,991, filed Apr. 26, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/042,141, filed Jan. 11, 2002, which is a continuation of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/726,643, filed Dec. 1, 2000, which is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/15187, filed Jun. 2, 2000, which claims the benefit of U.S. Provisional Application No. 60/137,725, filed Jun. 7, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/756,168, filed Jan. 9, 2001, which is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/19735, filed Jul. 20, 2000, which claims the benefit of U.S. Provisional Application No. 60/145,220, filed Jul. 23, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 10/060,255, filed Feb. 1, 2002, which is a continuation of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. Application No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/781,417, filed Feb. 13, 2001, which is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/22325, filed Aug. 16, 2000, which claims the benefit of U.S. Provisional Application No. 60/149,182, filed Aug. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/789,561, filed Feb. 22, 2001, which is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/24008, filed Aug. 31, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/152,315, filed Sep. 3, 1999, and 60/152,317, filed Sep. 3, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/800,729, filed Mar. 8, 2001, which is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/26013, filed Sep. 22, 2000, which claims the benefit of U.S. Provisional Application No. 60/155,709, filed Sep. 24, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of U.S. application Ser. No. 09/832,129, filed Apr. 11, 2001, which is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/28664, filed Oct. 17, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/163,085, filed Nov. 2, 1999, and 60/172,411, filed Dec. 17, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29363, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,139, filed Jun. 30, 2000, and 60/162,239, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29360, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,138, filed Jun. 30, 2000, and 60/162,211, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29362, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,131, filed Jun. 30, 2000, and 60/162,240, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29365, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,666, filed Jul. 21, 2000, and 60/162,237, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/29364, filed Oct. 25, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,134, filed Jun. 30, 2000, and 60/162,238, filed Oct. 29, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30040, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,130, filed Jun. 30, 2000, and 60/163,580, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30037, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,137, filed Jun. 30, 2000, and 60/163,577, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30045, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,133, filed Jun. 30, 2000, and 60/163,581, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30036, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,366, filed Jul. 27, 2000, and 60/163,576, filed Nov. 5, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30039, filed Nov. 1, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,367, filed Jul. 27, 2000, 60/195,296, filed Apr. 7, 2000, and 60/164,344, filed Nov. 9, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30654, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,142, filed Jul. 27, 2000, and 60/164,835, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30628, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,140, filed Jun. 30, 2000, and 60/164,744, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30653, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/221,193, filed Jul. 27, 2000, and 60/164,735, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30629, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/222,904, filed Aug. 3, 2000, and 60/164,825, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30679, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/224,007, filed Aug. 4, 2000, and 60/164,834, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30674, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,128, filed Jun. 30, 2000, and 60/164,750, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31162, filed Nov. 15, 2000; International Application No. PCT/US00/31162, filed Nov. 15, 2000 claims the benefit of U.S. Provisional Application Nos. 60/215,136, and 60/166,415, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/31282, filed Nov. 15, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/219,665, filed Jul. 21, 2000, and 60/166,414, filed Nov. 19, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US00/30657, filed Nov. 8, 2000, which claims the benefit of U.S. Provisional Application Nos. 60/215,132, filed Jun. 30, 2000, and 60/164,731, filed Nov. 12, 1999; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01396, filed Jan. 17, 2001; International Application No. PCT/US01/01396, filed Jan. 17, 2001 claims the benefit of U.S. Provisional Application Nos. 60/256,968, filed Dec. 21, 2000, and 60/226,280, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01387, filed Jan. 17, 2001; International Application No. PCT/US01/01387, filed Jan. 17, 2001 claims the benefit of U.S. Provisional Application Nos. 60/259,803, filed Jan. 5, 2001, and 60/226,380, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01567, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/228,084, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01431, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,968, filed Sep. 12, 2000; International Application No. PCT/US01/01431 is a continuation-in-part of U.S. application Ser. No. 09/915,582, filed Jul. 27, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01432, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/236,326, filed Sep. 29, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/00544, filed Jan. 9, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,211, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01435, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,282, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01386, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/232,104, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01565, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/234,210, filed Sep. 20, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01394, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,805, filed Jan. 5, 2001, and 60/226,278, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01434, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,678, filed Jan. 5, 2001, and 60/226,279, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01397, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/226,281, filed Aug. 18, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01385, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application No. 60/231,969, filed Sep. 12, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01384, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,516, filed Jan. 4, 2001, and 60/228,086, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US01/01383, filed Jan. 17, 2001, which claims the benefit of U.S. Provisional Application Nos. 60/259,804, filed Jan. 5, 2001, and 60/228,083, filed Aug. 28, 2000; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05064, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,444, filed Jul. 12, 2001, and 60/270,658, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 is a continuation-in-part of International Application No. PCT/US02/05301, filed Feb. 21, 2002, which claims the benefit of U.S. Provisional Application Nos. 60/304,417, filed Jul. 12, 2001, and 60/270,625, filed Feb. 23, 2001; U.S. application Ser. No. 10/100,683 claims the benefit of U.S. Provisional Application Nos. 60/304,121, filed Jul. 11, 2001, 60/295,869, filed Jun. 6, 2001, 60/325,209, filed Sep. 28, 2001, 60/311,085, filed Aug. 10, 2001, 60/330,629, filed Oct. 26, 2001, 60/331,046, filed Nov. 7, 2001, 60/358,554, filed Feb. 22, 2002, and 60/358,714, filed Feb. 25, 2002. This application is a continuation-in-part of U.S. application Ser. No. 11/968,925, filed Jan. 3, 2008, which is a divisional of Ser. No. 10/644,765, filed Aug. 21, 2003, which is a continuation of International Application No. PCT/US02/05301, filed Feb. 21, 2002, which in turn claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Application Nos. 60/270,625 and 60/304,417, filed Feb. 23, 2001 and Jul. 12, 2001, respectively. This application is a continuation-in-part of U.S. application Ser. No. 12/538,668, filed Aug. 10, 2009, which is a continuation of U.S. application Ser. No. 11/240,769, filed Oct. 3, 2005, which is a continuation application of U.S. application Ser. No. 09/997,131, filed Nov. 30, 2001, now abandoned, which is a continuation application of U.S. application Ser. No. 09/628,508, filed Jul. 28, 2000, now abandoned, which is a continuation-in-part of PCT International Application Serial No. PCT/US00/03062, filed Feb. 8, 2000, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Application No. 60/119,468 filed Feb. 10, 1999. This application is a continuation-in-part of U.S. application Ser. No. 11/777,133, filed Jul. 12, 2007, which is a continuation of U.S. application Ser. No. 11/229,769, filed Sep. 20, 2005, which is a continuation of U.S. application Ser. No. 10/233,453, filed Sep. 4, 2002, which is a divisional of U.S. application Ser. No. 09/489,847 filed Jan. 24, 2000, which is a continuation-in-part of International Application No. PCT/US99/17130 filed Jul. 29, 1999, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Applications Nos. 60/094,657, filed Jul. 30, 1998, 60/095,486, filed Aug. 5, 1998, 60/096,319, filed Aug. 12, 1998, 60/095,454, filed Aug. 6, 1998, and 60/095,455, filed Aug. 6, 1998. This application is a continuation-in-part of U.S. application Ser. No. 12/268,263, filed Nov. 10, 2008, which is a divisional of U.S. application Ser. No. 11/375,555, filed Mar. 15, 2006, which is a continuation-in-part of application Ser. No. 10/103,295, filed Mar. 22, 2002 (now U.S. Pat. No. 7,091,315, issued Aug. 15, 2006), which is a continuation-in-part of International Application No. PCT/US01/29871, filed Sep. 24, 2001, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/234,925, filed Sep. 25, 2000; application Ser. No. 10/103,295 is also a continuation-in-part of International Application No. PCT/US01/00911, filed Jan. 12, 2001 (now abandoned); application Ser. No. 10/103,295 is also a continuation-in-part of U.S. application Ser. No. 09/482,273, filed Jan. 13, 2000 (now U.S. Pat. No. 6,534,631, issued Mar. 18, 2003), which is a continuation-in-part of International Application No. PCT/US99/15849, filed Jul. 14, 1999, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 60/092,921, 60/092,922, and 60/092,956, all of which were filed on Jul. 15, 1998. This application is a continuation-in-part of U.S. application Ser. No. 11/760,578, filed Jun. 8, 2007, which is a continuation of U.S. application Ser. No. 10/886,642, filed Jul. 9, 2004, which is a continuation of U.S. application Ser. No. 10/050,873, filed Jan. 18, 2002, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 60/263,230, filed on Jan. 23, 2001, and 60/263,681, filed on Jan. 24, 2001; said U.S. application Ser. No. 10/050,873 is also a continuation-in-part of U.S. patent application Ser. No. 09/461,325, filed on Dec. 14, 1999, now U.S. Pat. No. 6,475,753, which is a continuation-in-part of International Patent Application No: PCT/US99/13418, filed on Jun. 15, 1999, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 60/089,507, 60/089,508, 60/089,509, 60/089,510, each of which was filed on Jun. 16, 1998, and 60/090,112 and 60/090,113, each of which was filed on Jun. 22, 1998. This application is a continuation-in-part of U.S. application Ser. No. 12/274,626, filed Nov. 20, 2008, which is a divisional of U.S. application Ser. No. 11/608,978, filed Dec. 11, 2006, which is a continuation of U.S. application Ser. No. 10/918,446, filed Aug. 16, 2004, which is a divisional of U.S. application Ser. No. 10/062,548, filed Feb. 5, 2002 (now U.S. Pat. No. 6,924,356, issued Aug. 2, 2005), which is a continuation of U.S. application Ser. No. 09/369,247, filed Aug. 5, 1999 (now U.S. Pat. No. 6,569,992, issued May 27, 2003), which is a continuation-in-part of International Application PCT/US99/02293, filed Feb. 4, 1999, which is a non-provisional of, and claims benefit under 35 U.S.C. §119(e) to, U.S. Provisional Applications 60/074,118, filed Feb. 9, 1998, 60/074,157, filed Feb. 9, 1998, 60/074,037, filed Feb. 9, 1998, 60/074,141, filed Feb. 9, 1998, and 60/074,341, filed Feb. 9, 1998. This application is a continuation-in-part of U.S. application Ser. No. 11/780,874, filed Jul. 20, 2007, which is a continuation of U.S. patent application Ser. No. 10/935,098, filed Sep. 8, 2004, which is a continuation of U.S. patent application Ser. No. 09/938,671, filed Aug. 27, 2001, which is a continuation of U.S. patent application Ser. No. 09/739,907, filed Dec. 20, 2000 (now abandoned), which is a continuation of U.S. patent application Ser. No. 09/348,457, filed Jul. 7, 1999 (now abandoned), which is a continuation-in-part of International Application No. PCT/US99/00108, filed Jan. 6, 1999, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Application Nos. 60/070,704, filed Jan. 7, 1998; 60/070,658, filed Jan. 7, 1998; 60/070,692, filed Jan. 7, 1998; and 60/070,657, filed Jan. 7, 1998. This application is a continuation-in-part of U.S. application Ser. No. 12/325,800, filed Dec. 1, 2008, which is a divisional of U.S. application Ser. No. 11/565,909, filed Dec. 1, 2006, which is a divisional of U.S. application Ser. No. 10/970,493, filed on Oct. 22, 2004 (now U.S. Pat. No. 7,163,797, issued Jan. 16, 2007), which is a continuation of U.S. application Ser. No. 10/047,021, filed on Jan. 17, 2002 (now abandoned), which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/262,066, filed on Jan. 18, 2001; U.S. application Ser. No. 10/047,021 is also a continuation-in-part of U.S. application Ser. No. 09/722,329, filed on Nov. 28, 2000 (now abandoned), which is a continuation of U.S. application Ser. No. 09/262,109, filed on Mar. 4, 1999 (now abandoned), which is a continuation-in-part of International Application PCT/US98/18360, filed on Sep. 3, 1998, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 60/057,626; 60/057,663; 60/057,669, filed on Sep. 5, 1997; 60/058,666; 60/058,667; 60/058,973; 60/058,974, filed on Sep. 12, 1997; and, 60/090,112, filed on Jun. 22, 1998. This application is a continuation-in-part of U.S. application Ser. No. 11/735,351, filed Apr. 13, 2007, which is a continuation of U.S. application Ser. No. 10/866,878, filed Jun. 15, 2004, which is a divisional of U.S. application Ser. No. 09/973,278, filed Oct. 10, 2001, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/239,899, filed Oct. 13, 2000; U.S. application Ser. No. 09/973,278 is also a continuation-in-part of U.S. application Ser. No. 09/227,357, filed Jan. 8, 1999, which is a continuation-in-part of International Application No. PCT/US98/13684, filed Jul. 7, 1998, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Applications: 60/051,926; 60/052,793; 60/051,925; 60/051,929; 60/052,803; 60/052,732; 60/051,931; 60/051,932; 60/051,916; 60/051,930; 60/051,918; 60/051,920; 60/052,733; 60/052,795; 60/051,919; 60/051,928 (each of which was filed Jul. 8, 1997); and; 60/055,722; 60/055,723; 60/055,948; 60/055,949; 60/055,953; 60/055,950; 60/055,947; 60/055,964; 60/056,360; 60/055,684; 60/055,984; 60/055,954 (each of which was filed Aug. 18, 1997); and; 60/058,785; 60/058,664; 60/058,660; 60/058,661 (each of which was filed Sep. 12, 1997). This application is a continuation-in-part of U.S. application Ser. No. 11/759,448, filed Jun. 7, 2007, which is a continuation of U.S. application Ser. No. 11/229,770, filed Sep. 20, 2005 (abandoned), which is a continuation of U.S. application Ser. No. 09/933,767, filed Aug. 22, 2001 (abandoned), which is a continuation-in-part of International Application No. PCT/US01/05614, filed Feb. 21, 2001, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Patent Application Ser. Nos. 60/184,836, filed Feb. 24, 2000 and 60/193,170, filed Mar. 29, 2000. Application Ser. No. 09/933,767 is a continuation-in-part of U.S. patent application Ser. No. 09/205,258, filed Dec. 4, 1998 (now U.S. Pat. No. 6,525,174), which is a continuation-in-part of International Patent Application No. PCT/US98/11422, filed Jun. 4, 1998, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Applications: 60/048,885; 60/049,375; 60/048,881; 60/048,880; 60/048,896; 60/049,020; 60/048,876; 60/048,895; 60/048,884; 60/048,894; 60/048,971; 60/048,964; 60/048,882; 60/048,899; 60/048,893; 60/048,900; 60/048,901; 60/048,892; 60/048,915; 60/049,019; 60/048,970; 60/048,972; 60/048,916; 60/049,373; 60/048,875; 60/049,374; 60/048,917; 60/048,949; 60/048,974; 60/048,883; 60/048,897; 60/048,898; 60/048,962; 60/048,963; 60/048,877; 60/048,878, all filed Jun. 6, 1997, and 60/057,645; 60/057,642; 60/057,668; 60/057,635; 60/057,627; 60/057,667; 60/057,666; 60/057,764; 60/057,643; 60/057,769; 60/057,763; 60/057,650; 60/057,584; 60/057,647; 60/057,661; 60/057,662; 60/057,646; 60/057,654; 60/057,651; 60/057,644; 60/057,765; 60/057,762; 60/057,775; 60/057,648; 60/057,774; 60/057,649; 60/057,770; 60/057,771; 60/057,761; 60/057,760; 60/057,776; 60/057,778; 60/057,629; 60/057,628; 60/057,777; 60/057,634, all filed Sep. 5, 1997, and 60/070,923, filed Dec. 18, 1997. This application in a continuation-in-part of U.S. application Ser. No. 12/324,825, filed Nov. 26, 2008, which is a continuation of U.S. application Ser. No. 11/226,657, filed Sep. 15, 2005, which is a divisional of U.S. application Ser. No. 10/062,831, filed Feb. 5, 2002, (now U.S. Pat. No. 7,001,992, issued Feb. 21, 2006), which is a divisional of U.S. application Ser. No. 09/690,454, filed Oct. 18, 2000 (now U.S. Pat. No. 6,531,447, issued Mar. 11, 2003), which is a continuation of U.S. application Ser. No. 09/189,144, filed Nov. 10, 1998 (now abandoned), which is a continuation-in-part of International Application No. PCT/US98/10868, filed May 28, 1998, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Applications: 60/044,039; 60/048,093; 60/048,190; 60/050,935; 60/048,101; 60/048,356 (each filed May 30, 1997); and 60/056,250; 60/056,296; 60/056,293 (each filed Aug. 29, 1997). This application in a continuation-in-part of U.S. application Ser. No. 11/801,040, filed May 7, 2007, which is a divisional of U.S. application Ser. No. 10/062,831, filed Feb. 5, 2002, which is a divisional of U.S. application Ser. No. 09/690,454, filed Oct. 18, 2000 (now U.S. Pat. No. 6,531,447, issued Mar. 11, 2003), which is a continuation of U.S. application Ser. No. 09/189,144 filed Nov. 10, 1998 (now abandoned), which is a continuation-in-part of International Application No. PCT/US98/10868, filed May 28, 1998, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Applications: 60/044,039; 60/048,093; 60/048,190; 60/050,935; 60/048,101; 60/048,356 (each filed May 30, 1997); and, 60/056,250; 60/056,296; 60/056,293 (each filed Aug. 29, 1997). This application in a continuation-in-part of U.S. application Ser. No. 11/744,695, filed May 4, 2007, which is a continuation of U.S. application Ser. No. 10/951,993 filed on Sep. 29, 2004, which is a divisional of U.S. application Ser. No. 10/058,993 filed on Jan. 30, 2002, which is a non-provisional of, and claims benefit under 35 U.S.C. §119(e) based on, U.S. Provisional Application No. 60/265,583 filed on Feb. 2, 2001; which is also a continuation-in-part, and claims priority under 35 U.S.C. §120, of U.S. application Ser. Nos. 09/852,659, 09/852,797, and 09/853,161 filed on May 11, 2001, each of which claim benefit under 35 U.S.C. §119(e) based on U.S. Provisional Application No. 60/265,583 filed on Feb. 2, 2001, and each of which is a continuation-in-part, and claim priority under 35 U.S.C. §120, of U.S. application Ser. No. 09/152,060 filed on Sep. 11, 1998, which is a continuation-in-part, and claims priority under 35 U.S.C. §120, of International Application PCT/US98/04858 filed on Mar. 12, 1998, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Applications 60/040,710 and 60/040,762 (filed on Mar. 14, 1997), 60/050,934, 60/048,100, 60/048,189 and 60/048,357 (filed on May 30, 1997), 60/048,970 (filed on Jun. 6, 1997), 60/057,765 (filed on Sep. 5, 1997), and 60/068,368 (filed on Dec. 19, 1997); and U.S. application Ser. No. 10/951,993 is also a continuation-in-part, and claims priority under 35 U.S.C. §120, of U.S. application Ser. No. 09/152,060 filed on Sep. 11, 1998, which is a continuation-in-part, and claims priority under 35 U.S.C. §120, of International Application PCT/US98/04858 filed on Mar. 12, 1998, which claims benefit under 35 U.S.C. §119(e) based on U.S. Provisional Applications 60/040,710 and 60/040,762 (filed on Mar. 14, 1997), 60/050,934, 60/048,100, 60/048,189 and 60/048,357 (filed on May 30, 1997), 60/048,970 (filed on Jun. 6, 1997), 60/057,765 (filed on Sep. 5, 1997), and 60/068,368 (filed on Dec. 19, 1997). This application is a continuation-in-part of U.S. application Ser. No. 11/745,580, filed May 8, 2007, which is a continuation of U.S. application Ser. No. 11/144,947, filed Jun. 6, 2005, which is a continuation of U.S. application Ser. No. 09/882,171, filed Jun. 18, 2001, which is a continuation application of U.S. application Ser. No. 09/809,391, filed Mar. 16, 2001, now abandoned, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/190,068, filed Mar. 17, 2000; U.S. application Ser. No. 11/144,947 is a continuation-in-part of U.S. application Ser. No. 10/164,861, filed Jun. 10, 2002, which is a divisional of U.S. application Ser. No. 09/149,476, filed Sep. 8, 1998, (now issued U.S. Pat. No. 6,420,526, issued on Jul. 16, 2002), which is a continuation-in-part of PCT International Application No. PCT/US98/04493, filed Mar. 6, 1998 which claims benefit under 35 U.S.C. §119(e) based on the following U.S. Provisional Applications: 60/040,162; 60/040,333; 60/038,621; 60/040,626; 60/040,334; 60/040,336; 60/040,163; (each filed Mar. 7, 1997); and, 60/047,600; 60/047,615; 60/047,597; 60/047,502; 60/047,633; 60/047,583; 60/047,617; 60/047,618; 60/047,503; 60/047,592; 60/047,581; 60/047,584; 60/047,500; 60/047,587; 60/047,492; 60/047,598; 60/047,613; 60/047,582; 60/047,596; 60/047,612; 60/047,632; 60/047,601, (each filed May 23, 1997); and, 60/043,580; 60/043,568; 60/043,314; 60/043,569; 60/043,311; 60/043,671; 60/043,674; 60/043,669; 60/043,312; 60/043,313; 60/043,672; 60/043,315, (each filed Apr. 11, 1997); and, 60/048,974, (filed Jun. 6, 1997); and, 60/056,886; 60/056,877; 60/056,889; 60/056,893; 60/056,630; 60/056,878; 60/056,662; 60/056,872; 60/056,882; 60/056,637; 60/056,903; 60/056,888; 60/056,879; 60/056,880; 60/056,894; 60/056,911; 60/056,636; 60/056,874; 60/056,910; 60/056,864; 60/056,631; 60/056,845, 60/056,892, (each filed Aug. 22, 1997); and 60/057,761, (filed Sep. 5, 1997); and, 60/047,595; 60/047,599; 60/047,588; 60/047,585; 60/047,586; 60/047,590; 60/047,594; 60/047,589; 60/047,593; 60/047,614, (each filed May 23, 1997); and, 60/043,578, 60/043,576 (each filed Apr. 11, 1997); and, 60/047,501, (filed May 23, 1997); and, 60/043,670, (filed Apr. 11, 1997); and, 60/056,632; 60/056,664; 60/056,876; 60/056,881; 60/056,909; 60/056,875; 60/056,862; 60/056,887; 60/056,908, (each filed Aug. 22, 1997); and, 60/048,964, (filed Jun. 6, 1997); and, 60/057,650, (filed Sep. 5, 1997); and, 60/056,884, (filed Aug. 22, 1997); and, 60/057,669, (filed Sep. 5, 1997); and, 60/049,610, (filed Jun. 13, 1997); and, 60/061,060, (filed Oct. 2, 1997); and, 60/051,926, (filed Jul. 8, 1997); and, 60/052,874, (filed Jul. 16, 1997); and, 60/058,785, (filed Sep. 12, 1997); and, 60/055,724, (filed Aug. 18, 1997); and, 60/040,161, (filed Mar. 7, 1997). This application is a continuation-in-part of U.S. application Ser. No. 12/264,040, filed Nov. 3, 2008, which is a divisional of U.S. application Ser. No. 11/246,999, filed Oct. 11, 2005, which is a divisional of U.S. application Ser. No. 09/984,130, filed Oct. 29, 2001 (now abandoned), which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/243,792, filed on Oct. 30, 2000, and which is also a continuation-in-part of U.S. application Ser. No. 09/836,353, filed Apr. 18, 2001, (now abandoned), which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/198,407, filed Apr. 19, 2000, and which is a continuation-in-part of PCT International Application No. PCT/US99/25031 (published in English), filed Oct. 27, 1999, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 60/105,971, filed Oct. 28, 1998. Each of the above referenced patents and patent applications is hereby incorporated by reference herein in its entirety.

REFERENCE TO SEQUENCE LISTING AS TEXT FILE

This application refers to a “Sequence Listing” listed below, which is provided as a text file. The text file contains a document entitled “PS960_SeqList.txt” (473,508 bytes, created Apr. 2, 2010), which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel proteins/polypeptides (such as human secreted proteins/polypeptides), and isolated nucleic acid molecules encoding said proteins/polypeptides, useful for detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders related to said proteins/polypeptides (relatedness may be by direct or indirect association, by cause, by consequence, or by effect on said diseases and disorders), such as immune, cardiovascular, cancer, and other proliferative disorders and diseases. Antibodies that bind these polypeptides are also encompassed by the present invention. Also encompassed by the invention are vectors, host cells, and recombinant and synthetic methods for producing said polynucleotides, polypeptides, and/or antibodies. The invention further encompasses screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further encompasses methods and compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating diseases, disorders, and/or conditions related to these novel human secreted proteins.

BACKGROUND OF THE INVENTION

Unlike bacterium, which exist as a single compartment surrounded by a membrane, human cells and other eukaryotes are subdivided by membranes into many functionally distinct compartments. Each membrane-bounded compartment, or organelle, contains different proteins essential for the function of the organelle. The cell uses “sorting signals,” which are amino acid motifs located within the protein, to target proteins to particular cellular organelles.

One type of sorting signal, called a signal sequence, a signal peptide, or a leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER). The ER separates the membrane-bounded proteins from all other types of proteins. Once localized to the ER, both groups of proteins can be further directed to another organelle called the Golgi apparatus. Here, the Golgi distributes the proteins to vesicles, including secretory vesicles, the cell membrane, lysosomes, and the other organelles.

Proteins targeted to the ER by a signal sequence can be released into the extracellular space as a secreted protein. For example, vesicles containing secreted proteins can fuse with the cell membrane and release their contents into the extracellular space—a process called exocytosis. Exocytosis can occur constitutively or after receipt of a triggering signal. In the latter case, the proteins are stored in secretory vesicles (or secretory granules) until exocytosis is triggered. Similarly, proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a “linker” holding the protein to the membrane.

Thus there exists a clear need for identifying and using novel secreted polynucleotides and polypeptides. Identification and sequencing of human genes is a major goal of modern scientific research. For example, by identifying genes and determining their sequences, scientists have been able to make large quantities of valuable human “gene products.” These include human insulin, interferon, Factor VIII, tumor necrosis factor, human growth hormone, tissue plasminogen activator, erythropoietin, and numerous other compounds. Thus, in light of the pervasive role of secreted proteins in human physiology, a need exists for identifying and characterizing novel human secreted proteins and the genes that encode them. This knowledge will allow one to detect, to treat, and to prevent medical diseases, disorders, and/or conditions by using secreted proteins or the genes that encode them. Additionally, knowledge of gene sequences can provide the key to treatment or cure of genetic diseases (such as muscular dystrophy and cystic fibrosis).

Immune-Related Polynucleotides and Polypeptides

The immune system is an intricate network of cells, tissues and soluble molecules that function to protect the body from invasion by foreign substances and pathogens. The major cells of the immune system are lymphocytes, including B cells and T cells, and myeloid cells, including basophils, eosinophils, neutrophils, mast cells, monocytes, macrophages and dendritic cells. In addition to these cellular components of the immune system, soluble molecules—such as antibodies, complement proteins, and cytokines—circulate in lymph and blood plasma, and play important roles in immunity.

The immune system can be subdivided into the acquired and innate immune systems. The cells of the innate immune system (e.g., neutrophils, eosinophils, basophils, mast cells) are not antigen specific and their action is not enhanced by repeated exposure to the same antigen. The cells of the acquired immune system (B and T cells) are antigen specific. Repeated exposure of B and T cells to an antigen results in improved immune responses (memory responses) produced by these cell types. The cells and products of the acquired immune system can recruit components of the innate system to mount a focused immune response. For a more extensive review of the immune system, see Fundamental Immunology, 4th edition, Ed. William Paul, Lippincott-Raven Pub. (1998).

An immune response is seldom carried out by a single cell type, but rather requires the coordinated efforts of several cell types. In order to coordinate an immune response, it is necessary that cells of the immune system communicate with each other and with other cells of the body. Communication between cells may be made by cell-cell contact, between membrane bound molecules on each cell, or by the interaction of soluble components of the immune system with cellular receptors. Signaling between cell types may have one or more of a variety of consequences, including activation, proliferation, differentiation, and apoptosis. Activation and differentiation of immune cells may result in the expression or secretion of polypeptides, or other molecules, which in turn affect the function of other cells and/or molecules of the immune system.

Molecules which stimulate or suppress immune system function are known as immunomodulators. These molecules, which include endogenous proteins (e.g., cytokines, cytokine receptors, and intracellular signal transduction molecules), molecules derived from microorganisms, and synthetic agents, may exert their modulatory effects at one or more stages of the immune response, such as antigen recognition, stimulation of cytokine production and release, and/or activation/differentiation of lymphocytes and myeloid cells. Immunomodulators may enhance (immunoprophylaxis, immunostimulation), restore (immunosubstitution, immunorestoration) or suppress (immunosuppression, immunodeviation) immunological functions or activities.

Immunomodulatory compounds have many important applications in clinical practice. For example, immunosuppressing agents (which attenuate or prevent unwanted immune responses) can be used to prevent tissue rejection during organ transplantation, to prevent Rh hemolytic disease of the newborn, or to treat autoimmune disorders. A mechanism of action common to many immunosuppressants is the inhibition of T cell activation and/or differentiation. Antilymphocyte antibodies have also been used to attenuate immune system functions. Currently-used immunosuppressive agents can produce a number of side effects which limit their use. Among the most serious secondary effects include kidney and liver toxicity, increased risk of infection, hyperglycemia, neoplasia, and osteoporosis (see, e.g., Freeman, Clin. Biochem. 24(1):9-14 (1991); Mitchison, Dig. Dis. 11(2):78-101 (1993)).

Immunostimulants, which enhance the activity of immune cells and molecules, comprise another class of immunomodulatory agents with important clinical applications. Such applications include, for example, the treatment of immunodeficiency disorders (e.g. AIDS and severe combined immunodeficiency), chronic infectious diseases (e.g. viral hepatitis, papillomavirus, and herpesvirus), and cancer. An important class of endogenous immunostimulants is the cytokines These soluble signaling molecules are produced by a number of cell types, and are critical to the regulation of the immune response. Immunostimulatory mechanisms can include proliferation, differentiation and/or activation of immune cells or progenitors of immune cells. For example, interleukin-2 (IL-2) binds to IL-2 receptors on T lymphocytes and induces proliferation and differentiation. Another cytokine, interferon alpha, stimulates the immune system through a variety of mechanisms, including activation of macrophages, T lymphocytes, and natural killer cells. Interferon alpha also induces the expression of antiviral proteins (see Chapter 50, The Pharmacological Basis of Therapeutics, 9^(th) Edition, Eds. Hardman, Limbird, Molinoff, Ruddon, and Gilman, McGraw Hill (1996)). Limitations of current immunostimulant therapies include anaphylaxis, pulmonary edema, and renal toxicity, to name a few.

The discovery of new human immune related polynucleotides, the polypeptides encoded by them, and antibodies that immunospecifically bind these polypeptides, satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, prevention and/or prognosis of disorders of the immune system, including, but not limited to, autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis, idiopathic thrombocytopenic purpura and multiple sclerosis), immunodeficiencies (e.g., X-linked agammaglobulinemia, severe combined immunodeficiency, Wiskott-Aldrich syndrome, and ataxia telangiectasia), chronic infections (e.g., HIV, viral hepatitis, and herpesvirus), and neoplastic disorders. See, e.g. “Immune Activity” section infra. Additionally, immune related molecules would be useful as agents to boost immune responsiveness to pathogens or to suppress immune reactions, for example as is necessary in conjunction with organ transplantation.

Cardiovascular-Related Polynucleotides and Polypeptides

The cardiovascular system is a component of a complex physiological network involved in maintaining the oxygen and nutrient supply to tissues of the body. The heart is the anatomical and functional centerpiece of the cardiovascular system. Weighing only 250-350 grams (less than a pound), the heart is one of our strongest and hardest working organs. It is composed of innervated muscle tissue with unique properties; e.g., it can pace itself in contraction. The main center of rhythm regulation is the sinoatrial (SA) node. Certain cardiac cells repeatedly fire impulses that trigger heart contractions. These autorhythmic cells have two important functions. One is to act as a pacemaker (set the pace for the entire heart), and the other is to form a conduction system, the route for conducting impulses throughout the heart muscle. This conduction system controls the pattern of blood flow through the heart.

The heart pumps at least five quarts of blood through a full circuit of the body every minute. The heart consists of two pumps, side by side. The pump on the right side moves blood to the lungs, where waste gases, such as carbon dioxide, are removed and oxygen is added. Freshly oxygenated blood returns to the pump on the left side, which moves it out into the rest of the body. Blood flows away from the heart to the lungs or to the rest of your body, though blood vessels called arteries. Arteries branch extensively, each branch become smaller, forming blood vessels called arterioles. Arterioles also become repeatedly smaller and smaller until they are tiny vessels called capillaries. Throughout the arteries and smaller vessels that stem from them, the blood delivers nutrients and oxygen to the tissues and picks up waste. This task is completed in the capillaries. As the blood moves on through the capillaries the blood vessels gradually become larger, eventually becoming veins. Veins ultimately carry blood back to the heart. The cycle then begins again.

Disorders of the cardiovascular system are many and varied, killing more Americans each year than any other category of disorders. For example, damage to the conduction system leads to arrhythmia, an irregular beating of the heart. If left untreated, the heart becomes unable to effectively pump blood, frequently leading to permanent heart damage and/or cardiac arrest.

One of the most prevalent conditions in industrialized countries today is atherosclerosis. Atherosclerosis is the buildup of fatty deposits in the intima of large and medium-sized arteries. The buildup of deposits narrowing of the arteries, reducing or potentially blocking the ability of blood to flow through the arteries. Untreated, atherosclerosis typically results in cardiac arrest and, frequently, death.

Clearly, the discovery of new human cardiovascular-associated polynucleotides, the polypeptides encoded by them, and antibodies that immunospecifically bind these polypeptides, satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, prevention and/or prognosis of caridovascular disorders.

Cardiovascular disorders include, but are not limited to, stroke, cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include, but are not limited to, aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.

Cardiovascular disorders also include, but are not limited to, heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include, but are not limited to, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve diseases include, but are not limited to, aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.

Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemias include, but are not limited to, coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include, but are not limited to, dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include, but are not limited to, arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include, but are not limited to, carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.

Embolisms include, but are not limited to, air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include, but are not limited to, coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemic disorders include, but are not limited to, cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes, but is not limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.

Cancer and Hyperproliferative-Related Polynucleotides and Polypeptides

Cancer and other hyperproliferative disorders are a diverse group of disorders and diseases sharing one characteristic in common; all result from uncontrolled cell proliferation. The human body is composed of many different cell types, e.g. liver cells, muscle cells, brain cells, etc. Normally, these cells grow and divide to produce more cells only as the body needs them (e.g. to regenerate blood cells or replace epithelial cells lining the stomach). Sometimes, however, cells begin to divide unchecked even though new cells are not needed. These extra cells accumulate and form a mass of tissue, called a tumor. Although each of the over 200 cell types in the body can potentially become cancerous, some cell types become cancerous at relatively high rates while many other cell types rarely become cancerous.

Tumors are either benign or malignant. Benign tumors are not cancerous; they can usually be removed, they do not spread to other parts of the body and, they rarely threaten life. Malignant tumors, however, are cancerous. Cells in malignant tumors can invade and damage nearby or distant tissues and organs. The spread of cancerous cells is called metastasis. Malignant (or metastatic) cells can invade adjacent organs by proliferating directly from the primary tumor. Additionally, malignant cells can also metastasize to distant organs by breaking away from the primary tumor, entering the bloodstream or lymphatic system, and settling down in a new organ or tissue to produce a secondary tumor. The origin of secondary tumors is established by comparing cells comprising these tumors to cells in the original (primary) tumor.

In contrast to solid organ cancers (such as cancer in the liver, lung, and brain) cancer can also develop in blood-forming cells. These cancers are referred to as leukemias or lymphomas. Leukemia refers to cancer of blood forming cells such as red blood cells, platelets, and plasma cells. Lymphomas are a subset of leukemias, primarily involving white blood cells, in which the cancerous cells originated in, or are associated with, the lymph system and lymph organs (e.g. T-lymphocytes in the lymph nodes, spleen, or thymus).

In 1999 over 1.1 million people were newly diagnosed with 23 different types of cancer. The vast majority of these cases (˜75%) involved cancers of the prostate, breast, lung, colon, or urinary tract, or non-Hodgkin's lymphoma. Among the most fatal cancers are pancreatic, liver, esophageal, lung, stomach, and brain cancers, having up to 96% mortality rates depending on the specific cancer. In all, some 23 different types of cancer are expected to kill over 86,000 people each year.

Most cancers are treated with one or a combination therapies consisting of surgery, radiation therapy, chemotherapy, hormone therapy, and/or biological therapy. These five therapeutic modes are either local or systemic treatment strategies. Local treatments affect cancer cells in the tumor and immediately adjacent areas (for example, surgical tumor removal is a local treatment as are most radiation treatments). In contrast, systemic treatments travel through the bloodstream, and reach cancer and other cells all over the body. Chemotherapy, hormone therapy, and biological therapy are examples of systemic treatments.

Whether systemic or local, it is often difficult or impossible to protect healthy cells from the harmful effects of cancer treatment; healthy cells and tissues are inevitably damaged in the process of treating the cancerous cells. Damage and disruption of the normal functioning of healthy cells and tissues often produces the undesirable side effects experienced by patients undergoing cancer treatment.

Recombinant polypeptides and polynucleotides derived from naturally occurring molecules, as well as antibodies specifically targeted to these molecules, used alone or in conjunction with other existing therapies, hold great promise as improved therapeutic agents for the treatment of neoplastic disorders. Currently, most biological therapy can be classified as immunotherapy because these treatments often use naturally occurring molecules to assist the body's immune system in fighting the disease or in protecting the body from side effects of other cancer treatment(s). Among the most commonly used compounds in biological therapies are proteins called cytokines (e.g. interferons, interleukins, and colony stimulating factors) and monoclonal antibodies (targeted to particular cancer cells). Side effects caused by these commonly used biological therapies range from flu-like symptoms (chills, fever, muscle aches, weakness, loss of appetite, nausea, vomiting, and diarrhea) to rashes, swelling, easy bruising, or bleeding.

The discovery of human secreted proteins associated with initiation, progression, characterization, and/or distinction of neoplastic diseases (including antibodies that immunospecifically bind these polypeptides), satisfies a need in the art by providing new compositions useful in the detection, prevention, diagnosis, treatment, prevention, prognosis, and treatment of hyperproliferative disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) corresponding to Gene No: 1.

FIG. 2 shows an analysis of the amino acid sequence (SEQ ID NO:2). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.

The data presented in FIG. 2 are also represented in tabular form in Table 8. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I-XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 2, and Table 8: “Res”: amino acid residue of SEQ ID NO:2 and FIG. 1; “Position”: position of the corresponding residue within SEQ ID NO:2 and FIG. 1; I: Alpha, Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman; III: Beta, Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V: Turn, Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil, Regions—Garnier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX: Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg; XI: Beta, Amphipathic Regions—Eisenberg; XII: Flexible Regions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV: Surface Probability Plot—Emini.

FIGS. 3A-3B show the Gene No: 5 nucleotide (SEQ ID NO:20) and deduced amino acid sequence (SEQ ID NO:21) corresponding to this gene.

FIG. 4 shows an analysis of the amino acid sequence (SEQ ID NO:21). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides

FIG. 5 shows the nucleotide (SEQ ID NO:102) and deduced amino acid sequence (SEQ ID NO:103) corresponding to Gene No: 14.

FIG. 6 shows an analysis of the amino acid sequence (SEQ ID NO:103). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.

FIGS. 7A-C show the nucleotide sequence (SEQ ID NO:134) and the deduced amino acid sequence (SEQ ID NO:135) of the HCEJQ69 cDNA (Gene No: 20).

FIG. 8 shows an analysis of the amino acid sequence (SEQ ID NO:135). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.

The data presented in FIG. 8 are also represented in tabular form in Table 9. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I-XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 8, and Table 9: “Res”: amino acid residue of SEQ ID NO:135 and FIGS. 7A-7C; “Position”: position of the corresponding residue within SEQ ID NO:135 and FIGS. 7A-7C; I: Alpha, Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman; III: Beta, Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V: Turn, Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil, Regions—Garnier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX: Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg; XI: Beta, Amphipathic Regions—Eisenberg; XII: Flexible Regions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV: Surface Probability Plot—Emini.

FIGS. 9A-B show the nucleotide (SEQ ID NO:159) and deduced amino acid sequence (SEQ ID NO:160) of this polypeptide.

FIG. 10 shows an analysis of the amino acid sequence (SEQ ID NO:160). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained.

FIGS. 11A-F show the nucleotide (SEQ ID NO:186) and deduced amino acid sequence (SEQ ID NO:187) of all. Predicted amino acids from about 1 to about 22 constitute the predicted signal peptide (amino acid residues from about 1 to about 22 in SEQ ID NO:187) and are represented by the underlined amino acid regions; amino acids from about 666 to about 682, and/or amino acids from about 1145 to about 1161 constitute the predicted transmembrane domains (amino acids from about 666 to about 682, and/or amino acids from about 1145 to about 1161 in SEQ ID NO:187) and are represented by the double underlined amino acids; and amino acids from about 64 to about 96 constitute the predicted immunoglobulin and major histocompatibility complex protein domain (amino acids from about 64 to about 96 in SEQ ID NO:187) and are represented by the bold amino acids.

FIGS. 12A-E show the regions of similarity between the amino acid sequences of the integrin alpha 11 subunit (all) protein (SEQ ID NO:187) and the human integrin alpha 1 subunit (SEQ ID NO:189).

FIG. 13 shows an analysis of the integrin alpha 11 subunit (all) amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.

SUMMARY OF THE INVENTION

The present invention relates to novel secreted proteins/polypeptides (such as human proteins/polypeptides). More specifically, isolated nucleic acid molecules are provided encoding novel secreted polypeptides. Novel polypeptides and antibodies that bind to these polypeptides are provided. Also provided are vectors, host cells, and recombinant and synthetic methods for producing human polynucleotides, polypeptides, and/or antibodies. The invention further relates to diagnostic and therapeutic methods useful for detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders related to said proteins/polypeptides (relatedness may be by direct or indirect association, or by cause, consequence, or effect on said diseases and disorders), such as immune, cardiovascular, cancer, and other proliferative disorders and diseases. The invention further relates to screening methods for identifying agonists and antagonists of polynucleotides and polypeptides of the invention. The present invention further relates to methods and/or compositions for inhibiting or enhancing the production and function of the polypeptides of the present invention.

DETAILED DESCRIPTION Polynucleotides and Polypeptides of the Invention Features of Protein Encoded by Gene No: 1

For purposes of this application, this gene and its corresponding translation product are known as the B7-H5 gene and B7-H5 protein. This protein is believed to reside as a cell-surface molecule, and the transmembrane domain of this protein is believed to embody the following preferred amino acid residues: IRVPVFNIVILLAGGF (SEQ ID NO:3). Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these peptides. The B7-H5 gene shares sequence homology with members of the B7 family of ligands (i.e., B7-1 (See Genbank Accession 507873)). These proteins and their corresponding receptors play vital roles in the growth, differentiation and death of T cells. For example, some members of this family (i.e., B7-H1) are involved in costimulation of the T cell response, as well as inducing increased cytokine production. Therefore, antagonists such as antibodies or small molecules directed against the B7-H5 gene are useful for treating T cell mediated immune system disorders. The gene encoding the disclosed cDNA is thought to reside on chromosome 6. Accordingly, polynucleotides related to this invention have uses, such as, for example, as a marker in linkage analysis for chromosome 6.

It has been discovered that this gene is expressed in activated neutrophils and activated T cells, and to a lesser extent in monocytes and heart tissue.

Preferred polypeptides of the present invention comprise, or alternatively consist of, one, two, three, four, five, six, seven or all seven of the immunogenic epitopes of the extracellular portion of the B7-H5 protein shown in SEQ ID NO:2 as residues: Leu-24 to Gln-35, Arg-59 to Pro-64, Glu-71 to His-78, Asp-89 to Gly-94, Pro-141 to Val-151, Thr-167 to Val-172, Ala-175 to Thr-180. Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these peptides.

In additional nonexclusive embodiments, polypeptides of the invention comprise, or alternatively consist of, one or more of the following amino acid sequences: 1.) The extracellular domain of the B7-H5 protein: MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIR DGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPP KEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTA DVSTPDSEINLTNVTDI (SEQ ID NO:4), 2.) The mature extracellular domain of the B7-H5 protein: EKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIIL EDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENS TQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDI (SEQ ID NO:5), and/or 3.) The leader sequence of the B7-H5 protein: MRKTRLWGLLWMLFVSELRAATKLTE (SEQ ID NO:6).

Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides.

Also preferred are polypeptides comprising, or alternatively consisting of, fragments of the mature extracellular portion of the B7-H5 protein demonstrating functional activity (SEQ ID NO:5). Polynucleotides encoding these polypeptides are also encompassed by the invention. By functional activity is meant, a polypeptide fragment capable of displaying one or more known functional activities associated with the full-length (complete) B7-H5 protein. Such functional activities include, but are not limited to, biological activity (e.g., T cell costimulatory activity, ability to bind ICOS, and ability to induce or inhibit cytokine production), antigenicity [ability to bind (or compete with a B7-H5 polypeptide for binding) to an anti-B7-H5 antibody], immunogenicity (ability to generate antibody which binds to a B7-H5 polypeptide), ability to form multimers with B7-H5 polypeptides of the invention, and ability to bind to a receptor or ligand for a B7-H5 polypeptide.

FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acid sequence (SEQ ID NO:2) corresponding to this gene.

FIG. 2 shows an analysis of the amino acid sequence (SEQ ID NO:2). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson-Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.

The data presented in FIG. 2 are also represented in tabular form in Table 8. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I-XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 2, and Table 8: “Res”: amino acid residue of SEQ ID NO:2 and FIG. 1; “Position”: position of the corresponding residue within SEQ ID NO:2 and FIG. 1; I: Alpha, Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman; III: Beta, Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V: Turn, Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil, Regions—Garnier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX: Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg; XI: Beta, Amphipathic Regions—Eisenberg; XII: Flexible Regions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV: Surface Probability Plot—Emini.

Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consisting of, one or more of the following regions: alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in FIG. 2 and/or Table 8, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 8 can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIG. 2, but may, as shown in Table 8, be represented or identified by using tabular representations of the data presented in FIG. 2. The DNA*STAR computer algorithm used to generate FIG. 2 (set on the original default parameters) was used to present the data in FIG. 2 in a tabular format (See Table 8). The tabular format of the data in FIG. 2 is used to easily determine specific boundaries of a preferred region.

The present invention is further directed to fragments of the polynucleotide sequences described herein. By a fragment of, for example, the polynucleotide sequence of a deposited cDNA or the nucleotide sequence shown in SEQ ID NO:1, is intended polynucleotide fragments at least about 15 nt, and more preferably at least about 20 nt, at least about 25 nt, still more preferably at least about 30 nt, at least about 35 nt, and even more preferably, at least about 40 nt in length, at least about 45 nt in length, at least about 50 nt in length, at least about 60 nt in length, at least about 70 nt in length, at least about 80 nt in length, at least about 90 nt in length, at least about 100 nt in length, at least about 125 nt in length, at least about 150 nt in length, at least about 175 nt in length, which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 200-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of a deposited cDNA or as shown in SEQ ID NO:1. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:1. In this context “about” includes the particularly recited size, an sizes larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, and from about 801 to about 860, of SEQ ID NO:1, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.

Preferred polypeptide fragments of the invention comprise, or alternatively consist of, the secreted protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. Particularly, N-terminal deletions of the polypeptide can be described by the general formula m-234 where m is an integer from 2 to 228, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:2. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: R-2 to P-234; K-3 to P-234; T-4 to P-234; R-5 to P-234; L-6 to P-234; W-7 to P-234; G-8 to P-234; L-9 to P-234; L-10 to P-234; W-11 to P-234; M-12 to P-234; L-13 to P-234; F-14 to P-234; V-15 to P-234; S-16 to P-234; E-17 to P-234; L-18 to P-234; R-19 to P-234; A-20 to P-234; A-21 to P-234; T-22 to P-234; K-23 to P-234; L-24 to P-234; T-25 to P-234; E-26 to P-234; E-27 to P-234; K-28 to P-234; Y-29 to P-234; E-30 to P-234; L-31 to P-234; K-32 to P-234; E-33 to P-234; G-34 to P-234; Q-35 to P-234; T-36 to P-234; L-37 to P-234; D-38 to P-234; V-39 to P-234; K-40 to P-234; C-41 to P-234; D-42 to P-234; Y-43 to P-234; T-44 to P-234; L-45 to P-234; E-46 to P-234; K-47 to P-234; F-48 to P-234; A-49 to P-234; 5-50 to P-234; 5-51 to P-234; Q-52 to P-234; K-53 to P-234; A-54 to P-234; W-55 to P-234; Q-56 to P-234; 1-57 to P-234; I-58 to P-234; R-59 to P-234; D-60 to P-234; G-61 to P-234; E-62 to P-234; M-63 to P-234; P-64 to P-234; K-65 to P-234; T-66 to P-234; L-67 to P-234; A-68 to P-234; C-69 to P-234; T-70 to P-234; E-71 to P-234; R-72 to P-234; P-73 to P-234; S-74 to P-234; K-75 to P-234; N-76 to P-234; S-77 to P-234; H-78 to P-234; P-79 to P-234; V-80 to P-234; Q-81 to P-234; V-82 to P-234; G-83 to P-234; R-84 to P-234; 1-85 to P-234; 1-86 to P-234; L-87 to P-234; E-88 to P-234; D-89 to P-234; Y-90 to P-234; H-91 to P-234; D-92 to P-234; H-93 to P-234; G-94 to P-234; L-95 to P-234; L-96 to P-234; R-97 to P-234; V-98 to P-234; R-99 to P-234; M-100 to P-234; V-101 to P-234; N-102 to P-234; L-103 to P-234; Q-104 to P-234; V-105 to P-234; E-106 to P-234; D-107 to P-234; S-108 to P-234; G-109 to P-234; L-110 to P-234; Y-111 to P-234; Q-112 to P-234; C-113 to P-234; V-114 to P-234; 1-115 to P-234; Y-116 to P-234; Q-117 to P-234; P-118 to P-234; P-119 to P-234; K-120 to P-234; E-121 to P-234; P-122 to P-234; H-123 to P-234; M-124 to P-234; L-125 to P-234; F-126 to P-234; D-127 to P-234; R-128 to P-234; 1-129 to P-234; R-130 to P-234; L-131 to P-234; V-132 to P-234; V-133 to P-234; T-134 to P-234; K-135 to P-234; G-136 to P-234; F-137 to P-234; S-138 to P-234; G-139 to P-234; T-140 to P-234; P-141 to P-234; G-142 to P-234; S-143 to P-234; N-144 to P-234; E-145 to P-234; N-146 to P-234; S-147 to P-234; T-148 to P-234; Q-149 to P-234; N-150 to P-234; V-151 to P-234; Y-152 to P-234; K-153 to P-234; 1-154 to P-234; P-155 to P-234; P-156 to P-234; T-157 to P-234; T-158 to P-234; T-159 to P-234; K-160 to P-234; A-161 to P-234; L-162 to P-234; C-163 to P-234; P-164 to P-234; L-165 to P-234; Y-166 to P-234; T-167 to P-234; S-168 to P-234; P-169 to P-234; R-170 to P-234; T-171 to P-234; V-172 to P-234; T-173 to P-234; Q-174 to P-234; A-175 to P-234; P-176 to P-234; P-177 to P-234; K-178 to P-234; S-179 to P-234; T-180 to P-234; A-181 to P-234; D-182 to P-234; V-183 to P-234; S-184 to P-234; T-185 to P-234; P-186 to P-234; D-187 to P-234; S-188 to P-234; E-189 to P-234; 1-190 to P-234; N-191 to P-234; L-192 to P-234; T-193 to P-234; N-194 to P-234; V-195 to P-234; T-196 to P-234; D-197 to P-234; 1-198 to P-234; 1-199 to P-234; R-200 to P-234; V-201 to P-234; P-202 to P-234; V-203 to P-234; F-204 to P-234; N-205 to P-234; 1-206 to P-234; V-207 to P-234; 1-208 to P-234; L-209 to P-234; L-210 to P-234; A-211 to P-234; G-212 to P-234; G-213 to P-234; F-214 to P-234; L-215 to P-234; S-216 to P-234; K-217 to P-234; S-218 to P-234; L-219 to P-234; V-220 to P-234; F-221 to P-234; S-222 to P-234; V-223 to P-234; L-224 to P-234; F-225 to P-234; A-226 to P-234; V-227 to P-234; T-228 to P-234; and/or L-229 to P-234 of SEQ ID NO:2. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Additionally, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the following group of C-terminal deletions: M-1 to V-233; M-1 to F-232; M-1 to S-231; M-1 to R-230; M-1 to L-229; M-1 to T-228; M-1 to V-227; M-1 to A-226; M-1 to F-225; M-1 to L-224; M-1 to V-223; M-1 to S-222; M-1 to F-221; M-1 to V-220; M-1 to L-219; M-1 to S-218; M-1 to K-217; M-1 to S-216; M-1 to L-215; M-1 to F-214; M-1 to G-213; M-1 to G-212; M-1 to A-211; M-1 to L-210; M-1 to L-209; M-1 to I-208; M-1 to V-207; M-1 to I-206; M-1 to N-205; M-1 to F-204; M-1 to V-203; M-1 to P-202; M-1 to V-201; M-1 to R-200; M-1 to I-199; M-1 to I-198; M-1 to D-197; M-1 to T-196; M-1 to V-195; M-1 to N-194; M-1 to T-193; M-1 to L-192; M-1 to N-191; M-1 to I-190; M-1 to E-189; M-1 to S-188; M-1 to D-187; M-1 to P-186; M-1 to T-185; M-1 to S-184; M-1 to V-183; M-1 to D-182; M-1 to A-181; M-1 to T-180; M-1 to S-179; M-1 to K-178; M-1 to P-177; M-1 to P-176; M-1 to A-175; M-1 to Q-174; M-1 to T-173; M-1 to V-172; M-1 to T-171; M-1 to R-170; M-1 to P-169; M-1 to S-168; M-1 to T-167; M-1 to Y-166; M-1 to L-165; M-1 to P-164; M-1 to C-163; M-1 to L-162; M-1 to A-161; M-1 to K-160; M-1 to T-159; M-1 to T-158; M-1 to T-157; M-1 to P-156; M-1 to P-155; M-1 to I-154; M-1 to K-153; M-1 to Y-152; M-1 to V-151; M-1 to N-150; M-1 to Q-149; M-1 to T-148; M-1 to S-147; M-1 to N-146; M-1 to E-145; M-1 to N-144; M-1 to S-143; M-1 to G-142; M-1 to P-141; M-1 to T-140; M-1 to G-139; M-1 to S-138; M-1 to F-137; M-1 to G-136; M-1 to K-135; M-1 to T-134; M-1 to V-133; M-1 to V-132; M-1 to L-131; M-1 to R-130; M-1 to I-129; M-1 to R-128; M-1 to D-127; M-1 to F-126; M-1 to L-125; M-1 to M-124; M-1 to H-123; M-1 to P-122; M-1 to E-121; M-1 to K-120; M-1 to P-119; M-1 to P-118; M-1 to Q-117; M-1 to Y-116; M-1 to I-115; M-1 to V-114; M-1 to C-113; M-1 to Q-112; M-1 to Y-111; M-1 to L-110; M-1 to G-109; M-1 to S-108; M-1 to D-107; M-1 to E-106; M-1 to V-105; M-1 to Q-104; M-1 to L-103; M-1 to N-102; M-1 to V-101; M-1 to M-100; M-1 to R-99; M-1 to V-98; M-1 to R-97; M-1 to L-96; M-1 to L-95; M-1 to G-94; M-1 to H-93; M-1 to D-92; M-1 to H-91; M-1 to Y-90; M-1 to D-89; M-1 to E-88; M-1 to L-87; M-1 to I-86; M-1 to I-85; M-1 to R-84; M-1 to G-83; M-1 to V-82; M-1 to Q-81; M-1 to V-80; M-1 to P-79; M-1 to H-78; M-1 to S-77; M-1 to N-76; M-1 to K-75; M-1 to S-74; M-1 to P-73; M-1 to R-72; M-1 to E-71; M-1 to T-70; M-1 to C-69; M-1 to A-68; M-1 to L-67; M-1 to T-66; M-1 to K-65; M-1 to P-64; M-1 to M-63; M-1 to E-62; M-1 to G-61; M-1 to D-60; M-1 to R-59; M-1 to I-58; M-1 to I-57; M-1 to Q-56; M-1 to W-55; M-1 to A-54; M-1 to K-53; M-1 to Q-52; M-1 to S-51; M-1 to S-50; M-1 to A-49; M-1 to F-48; M-1 to K-47; M-1 to E-46; M-1 to L-45; M-1 to T-44; M-1 to Y-43; M-1 to D-42; M-1 to C-41; M-1 to K-40; M-1 to V-39; M-1 to D-38; M-1 to L-37; M-1 to T-36; M-1 to Q-35; M-1 to G-34; M-1 to E-33; M-1 to K-32; M-1 to L-31; M-1 to E-30; M-1 to Y-29; M-1 to K-28; M-1 to E-27; M-1 to E-26; M-1 to T-25; M-1 to L-24; M-1 to K-23; M-1 to T-22; M-1 to A-21; M-1 to A-20; M-1 to R-19; M-1 to L-18; M-1 to E-17; M-1 to 5-16; M-1 to V-15; M-1 to F-14; M-1 to L-13; M-1 to M-12; M-1 to W-11; M-1 to L-10; M-1 to L-9; M-1 to G-8; and/or M-1 to W-7; of SEQ ID NO:2. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein (e.g., ability to inhibit the Mixed Lymphocyte Reaction), other functional activities (e.g., biological activities, ability to multimerize, ability to bind ligand, ability to generate antibodies, ability to bind antibodies) may still be retained. For example, the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a polypeptide with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response. Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in FIG. 1 (SEQ ID NO:2), as described by the general formula 1-n, where n is an integer from 6 to 228, where n corresponds to the position of the amino acid residue identified in SEQ ID NO:2.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group of N-terminal deletions of the mature extracellular portion of the B7-H5 protein (SEQ ID NO:5): K-28 to 1-198; Y-29 to I-198; E-30 to I-198; L-31 to I-198; K-32 to I-198; E-33 to I-198; G-34 to I-198; Q-35 to I-198; T-36 to I-198; L-37 to I-198; D-38 to I-198; V-39 to I-198; K-40 to I-198; C-41 to I-198; D-42 to I-198; Y-43 to I-198; T-44 to I-198; L-45 to I-198; E-46 to I-198; K-47 to I-198; F-48 to I-198; A-49 to I-198; 5-50 to I-198; 5-51 to I-198; Q-52 to I-198; K-53 to I-198; A-54 to I-198; W-55 to I-198; Q-56 to I-198; 1-57 to I-198; 1-58 to I-198; R-59 to I-198; D-60 to I-198; G-61 to I-198; E-62 to I-198; M-63 to I-198; P-64 to I-198; K-65 to I-198; T-66 to I-198; L-67 to I-198; A-68 to I-198; C-69 to I-198; T-70 to I-198; E-71 to I-198; R-72 to I-198; P-73 to I-198; S-74 to I-198; K-75 to I-198; N-76 to I-198; S-77 to I-198; H-78 to I-198; P-79 to I-198; V-80 to I-198; Q-81 to I-198; V-82 to I-198; G-83 to I-198; R-84 to I-198; 1-85 to I-198; 1-86 to I-198; L-87 to I-198; E-88 to I-198; D-89 to I-198; Y-90 to I-198; H-91 to I-198; D-92 to I-198; H-93 to I-198; G-94 to I-198; L-95 to I-198; L-96 to I-198; R-97 to I-198; V-98 to I-198; R-99 to I-198; M-100 to I-198; V-101 to 1-198; N-102 to I-198; L-103 to I-198; Q-104 to I-198; V-105 to I-198; E-106 to I-198; D-107 to I-198; S-108 to I-198; G-109 to I-198; L-110 to I-198; Y-111 to I-198; Q-112 to I-198; C-113 to I-198; V-114 to I-198; I-115 to I-198; Y-116 to I-198; Q-117 to I-198; P-118 to I-198; P-119 to I-198; K-120 to I-198; E-121 to I-198; P-122 to I-198; H-123 to I-198; M-124 to I-198; L-125 to I-198; F-126 to I-198; D-127 to I-198; R-128 to I-198; 1-129 to I-198; R-130 to I-198; L-131 to I-198; V-132 to I-198; V-133 to I-198; T-134 to I-198; K-135 to I-198; G-136 to I-198; F-137 to I-198; S-138 to I-198; G-139 to I-198; T-140 to I-198; P-141 to I-198; G-142 to I-198; S-143 to I-198; N-144 to I-198; E-145 to I-198; N-146 to I-198; S-147 to I-198; T-148 to I-198; Q-149 to I-198; N-150 to I-198; V-151 to I-198; Y-152 to I-198; K-153 to I-198; 1-154 to I-198; P-155 to I-198; P-156 to I-198; T-157 to I-198; T-158 to I-198; T-159 to I-198; K-160 to I-198; A-161 to I-198; L-162 to I-198; C-163 to I-198; P-164 to I-198; L-165 to I-198; Y-166 to I-198; T-167 to I-198; S-168 to I-198; P-169 to I-198; R-170 to I-198; T-171 to I-198; V-172 to I-198; T-173 to I-198; Q-174 to I-198; A-175 to I-198; P-176 to I-198; P-177 to I-198; K-178 to I-198; S-179 to I-198; T-180 to I-198; A-181 to I-198; D-182 to I-198; V-183 to I-198; S-184 to I-198; T-185 to I-198; P-186 to I-198; D-187 to I-198; S-188 to I-198; E-189 to I-198; 1-190 to I-198; N-191 to I-198; L-192 to I-198; T-193 to I-198; and/or K-23 to A-29 of SEQ ID NO:2. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Additionally, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group of C-terminal deletions of the mature extracellular portion of the B7-H5 protein (SEQ ID NO:5): E-27 to D-197; E-27 to T-196; E-27 to V-195; E-27 to N-194; E-27 to T-193; E-27 to L-192; E-27 to N-191; E-27 to I-190; E-27 to E-189; E-27 to S-188; E-27 to D-187; E-27 to P-186; E-27 to T-185; E-27 to S-184; E-27 to V-183; E-27 to D-182; E-27 to A-181; E-27 to T-180; E-27 to S-179; E-27 to K-178; E-27 to P-177; E-27 to P-176; E-27 to A-175; E-27 to Q-174; E-27 to T-173; E-27 to V-172; E-27 to T-171; E-27 to R-170; E-27 to P-169; E-27 to S-168; E-27 to T-167; E-27 to Y-166; E-27 to L-165; E-27 to P-164; E-27 to C-163; E-27 to L-162; E-27 to A-161; E-27 to K-160; E-27 to T-159; E-27 to T-158; E-27 to T-157; E-27 to P-156; E-27 to P-155; E-27 to I-154; E-27 to K-153; E-27 to Y-152; E-27 to V-151; E-27 to N-150; E-27 to Q-149; E-27 to T-148; E-27 to S-147; E-27 to N-146; E-27 to E-145; E-27 to N-144; E-27 to S-143; E-27 to G-142; E-27 to P-141; E-27 to T-140; E-27 to G-139; E-27 to S-138; E-27 to F-137; E-27 to G-136; E-27 to K-135; E-27 to T-134; E-27 to V-133; E-27 to V-132; E-27 to L-131; E-27 to R-130; E-27 to I-129; E-27 to R-128; E-27 to D-127; E-27 to F-126; E-27 to L-125; E-27 to M-124; E-27 to H-123; E-27 to P-122; E-27 to E-121; E-27 to K-120; E-27 to P-119; E-27 to P-118; E-27 to Q-117; E-27 to Y-116; E-27 to I-115; E-27 to V-114; E-27 to C-113; E-27 to Q-112; E-27 to Y-111; E-27 to L-110; E-27 to G-109; E-27 to S-108; E-27 to D-107; E-27 to E-106; E-27 to V-105; E-27 to Q-104; E-27 to L-103; E-27 to N-102; E-27 to V-101; E-27 to M-100; E-27 to R-99; E-27 to V-98; E-27 to R-97; E-27 to L-96; E-27 to L-95; E-27 to G-94; E-27 to H-93; E-27 to D-92; E-27 to H-91; E-27 to Y-90; E-27 to D-89; E-27 to E-88; E-27 to L-87; E-27 to I-86; E-27 to I-85; E-27 to R-84; E-27 to G-83; E-27 to V-82; E-27 to Q-81; E-27 to V-80; E-27 to P-79; E-27 to H-78; E-27 to S-77; E-27 to N-76; E-27 to K-75; E-27 to S-74; E-27 to P-73; E-27 to R-72; E-27 to E-71; E-27 to T-70; E-27 to C-69; E-27 to A-68; E-27 to L-67; E-27 to T-66; E-27 to K-65; E-27 to P-64; E-27 to M-63; E-27 to E-62; E-27 to G-61; E-27 to D-60; E-27 to R-59; E-27 to I-58; E-27 to I-57; E-27 to Q-56; E-27 to W-55; E-27 to A-54; E-27 to K-53; E-27 to Q-52; E-27 to S-51; E-27 to S-50; E-27 to A-49; E-27 to F-48; E-27 to K-47; E-27 to E-46; E-27 to L-45; E-27 to T-44; E-27 to Y-43; E-27 to D-42; E-27 to C-41; E-27 to K-40; E-27 to V-39; E-27 to D-38; E-27 to L-37; E-27 to T-36; E-27 to Q-35; E-27 to G-34; and/or E-27 to E-33 of SEQ ID NO:2. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

In addition, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides comprising, or alternatively consisting of, one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of SEQ ID NO:2, where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The present invention is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein as m-n. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N- and C-terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also included are polynucleotide sequences encoding a polypeptide consisting of a portion of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit Nos. 97903 and/or 209049, where this portion excludes any integer of amino acid residues from 1 to about 228 amino acids from the amino terminus of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit Nos. 97903 and/or 209049, or any integer of amino acid residues from 1 to about 228 amino acids from the carboxy terminus, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit Nos. 97903 and/or 209049. Polypeptides encoded by these polynucleotides also are encompassed by the invention.

As described herein or otherwise known in the art, the polynucleotides of the invention have uses that include, but are not limited to, serving as probes or primers in chromosome identification, chromosome mapping, and linkage analysis.

Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of immune system tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, diseases and/or disorders involving immune system activation, stimulation and/or surveillance, particularly involving T cells and/or neutrophils. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). Particularly contemplated are the use of antibodies directed against the extracellular portion of this protein which act as antagonists for the activity of the B7-H5 protein. Such antagonistic antibodies would be useful for the prevention and/or inhibition of such biological activites as are disclosed herein (e.g. T cell modulated activities).

For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in immune cells (e.g., T-cells, neutrophils), and the homology to members of the B7 family of ligands, indicates that the polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis, detection and/or treatment of diseases and/or disorders involving immune system activation, stimulation and/or surveillance, particularly as relating to T cells and/or neutrophils. In particular, the translation product of the B7-H5 gene may be involved in the costimulation of T cells, binding to ICOS, and/or may play a role in modulation of the expression of particular cytokines. Representative uses are also described in the “Immune Activity” section below and elsewhere herein.

More generally, the tissue distribution in immun system cells indicates that this gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:1 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 888 of SEQ ID NO:1, b is an integer of 15 to 902, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:1, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 2

This gene is expressed primarily in placenta and several tumors of various tissue origin and to a lesser extent in normal tissues including liver, lung, brain, and skin,

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of cancers of all kinds. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the central nervous system, respiratory system and skin, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues and cell types (e.g., liver, lung, brain and other tissues of the nervous system, and skin, and cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The high expression of this gene in multiple tumors indicates that the protein product of the clone may be involved in cell growth control and therefore would be useful for treatment of certain cancers. Likewise molecules developed to block the activity of the protein product of this clone could be used to block its potential role in tumor growth promotion.

This gene is believed to reside on chromosome 6. Polynucleotides related to this gene are believed, therefore, to be useful in linkage analysis for chromosome 6.

Preferred epitopes include those comprising a sequence shown in SEQ ID NO:8 as residues: Gln-37 to Gln-43, Cys-51 to Cys-65.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:7 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1909 of SEQ ID NO:7, b is an integer of 15 to 1923, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:7, and where the b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 3

The translation product of this gene shares sequence homology with CpG islands genes which are short stretches of DNA containing a high density of non-methylated CpG dinucleotides, predominantly associated with coding regions. As CpG islands overlap with approximately 60% of human genes, the CpG island library can be used to isolate full-length cDNAs and to place genes on genomic maps. The translation product also shares distant homology with the A33 protein, which is a transmembrane protein and a member of the immunoglobulin superfamily.

This gene is expressed primarily in the testes and to a lesser extent in the lung, tonsils, placenta, and rhabdomyosarcoma.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, diseases related to the testes, lung, tonsils, placenta, and tumors. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the diseases related to the testes, lung, tonsils, placenta, and tumors, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., testes and other reproductive tissue, lung, tonsils, placenta, and striated muscle, and cancerous and wounded tissues) or bodily fluids (e.g., seminal fluid, serum, plasma, urine, synovial fluid or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred epitopes include those comprising a sequence shown in SEQ ID NO:10 as residues: Met-1 to His-7.

The tissue distribution indicates that polynucleotides and polypeptides corresponding to the gene are useful for diagnosis and treatment of diseases related to the testes, lung, tonsils, placenta, and tumors. More specifically, the tissue distribution indicates that the protein product of this clone is useful for the treatment and diagnosis of conditions concerning proper testicular function (e.g. endocrine function, sperm maturation). Therefore, this gene product is useful in the treatment of male infertility and/or impotence. This gene product is also useful in assays designed to identify binding agents as such agents (antagonists) are useful as male contraceptive agents. Similarly, the protein is believed to by useful in the treatment and/or diagnosis of testicular cancer. The testes are also a site of active gene expression of transcripts that may be expressed, particularly at low levels, in other tissues of the body. Therefore, this gene product may be expressed in other specific tissues or organs where it may play related functional roles in other processes, such as hematopoiesis, inflammation, bone formation, and kidney function, to name a few possible target indications. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Features of Protein Encoded by Gene No: 4

When tested against Jurkat T cells, supernatants removed from cells containing this gene activated the GAS assay. Thus, it is likely that this gene activates T cells through the Jak-STAT signal transduction pathway. The gamma activating sequence (GAS) is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.

A preferred polypeptide variant of the invention comprises the following amino acid sequence: MARGSLRRLLRLLVLGLWLALLRSVAGEQAPGTAPCSRGSSWSADLDKCMDCSTSCPLPA ALAHPWGRSEPDLRAGAAFWLFGLETMPQEREVHHPHRGDRRRGLPSCGADPVTMCPLP AGARPLIIHSSILEPVSASQTRREPSSSNHKGGGGR (SEQ ID NO:19). Polynucleotides encoding these polypeptides are also provided.

The gene encoding the disclosed cDNA is believed to reside on chromosome 16. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 16. This gene is expressed primarily in tumor growth factor or lipopolysaccharide treated bone marrow stroma, epithelioid sarcoma, umbilical vein endothelial cells, in keratinocytes, and to a lesser extent, in other tissues including endothelial cells and placenta.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, hematopoiesis or immune disorders including integumentary or vascular disorders, particularly impaired wound healing and autoimmune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the skin and/or hematopoietic, integumentary, or immune systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g. hematopoietic, immune, integumentary, endothelial, and/or cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and/or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Preferred polypeptides of the present invention comprise immunogenic epitopes including those comprising a sequence shown in SEQ ID NOs: 14 and 12 as residues: Pro-35 to Trp-42, Ala-53 to Asp-62, Pro-65 to Asp-72, Thr-86 to Phe-93, Ile-97 to Glu-103, and Arg-103 to Pro-113. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in tumor growth factor or lipopolysaccharide treated bone marrow stroma, epithelioid sarcoma, and umbilical vein endothelial cells; and activation of the Jak-Stat promoter element in immune cells, specifically Jurkat T-cells, indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis, prevention, and/or treatment of a variety of immune system disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g., by boosting immune responses).

Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, scleroderma and tissues.

The tissue distribution in keratinocytes indicates that the protein products of this gene are useful for the treatment of wound healing deficiency and skin disorders, including congenital disorders (i.e. nevi, moles, freckles, Mongolian spots, hemangiomas, port-wine syndrome), integumentary tumors (i.e. keratoses, Bowen's disease, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease, mycosis fungoides, and Kaposi's sarcoma), injuries and inflammation of the skin (i.e. wounds, rashes, prickly heat disorder, psoriasis, dermatitis), atherosclerosis, uticaria, eczema, photosensitivity, autoimmune disorders (i.e. lupus erythematosus, vitiligo, dermatomyositis, morphea, scleroderma, pemphigoid, and pemphigus), keloids, striae, erythema, petechiae, purpura, and xanthelasma. Moreover, such disorders may predispose increased susceptibility to viral and bacterial infections of the skin (i.e. cold sores, warts, chickenpox, molluscum contagiosum, herpes zoster, boils, cellulitis, erysipelas, impetigo, tinea, althletes foot, and ringworm). Moreover, expression within endothelial cells and pancreatic tissues indicates that the protein product of this gene may be useful in the treatment and/or prevention of a variety of vascular disorders, particularly those involving highly vascularized tissues, which include, but are not limited to, embolism, aneurysm, stroke, atherosclerosis, and miscrovascular disease. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. The secreted protein can be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions and as nutritional supplements. It may also have a very wide range of biological activities although no evidence for any is provided in the specification. Typical of these are cytokine, cell proliferation/differentiation modulating activity or induction of other cytokines; immunostimulating/immunosuppressant activities (e.g., for treating human immunodeficiency-virus infection, cancer, autoimmune diseases and allergy); regulation of haematopoiesis (e.g., for treating anaemia or as adjunct to chemotherapy); stimulation of growth of bone, cartilage, tendons, ligaments and/or nerves (e.g., for treating wounds, stimulation of follicle stimulating hormone (for control of fertility); chemotactic and chemokinetic activities (e.g., for treating infections, tumours); haemostatic or thrombolytic activity (e.g., for treating haemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g., for treating septic shock, Crohn's disease); as antimicrobials; for treating psoriasis or other hyperproliferative disease; for regulation of metabolism, behaviour, and many others. Also contemplated is the use of the corresponding nucleic acid in gene therapy procedures. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NOs: 11 and 13 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1027 of SEQ ID NO:11 or 1 to 1038 of SEQ ID NO:13, b is an integer of 15 to 1041 of SEQ ID NO:11 or 15 to 1052 of SEQ ID NO:13, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NOs: 11 and 13, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 5

In a specific embodiment, polypeptides of the invention, comprise or alternatively consist of, one or more of the following amino acid sequences: TTILRTCTIVCFYYWFNGVMVLLFFLDRNLLTFNQASIMPFSNTDFLHCLSFKKKLMLLRYI FYVVLTGPTLSLKGDENQIKNLFT (SEQ ID NO:24), IVCFYYWFNGVMVLLFFLDRNLL (SEQ ID NO:25), and/or LLRYIFYVVLTGPTLSLKGDENQI (SEQ ID NO:26). Polynucleotides encoding these polypeptides are also encompassed by the invention as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also preferred are polypeptides, comprising or alternatively consisting of, the mature polypeptide which is predicted to consist of residues: PTCYSRMRALSQEITRDFNLLQVSEPSEPCVRYLPRLYLDIHNYCVLDKLRDFVASPPCWK VAQVDSLKDKARKLYTIMNSFCRRDLVFLLDDCNALEYPIPVTTVLPDRQR (SEQ ID NO:27) of the foregoing sequence (SEQ ID NO:21), and biologically active fragments of the mature polypeptide (e.g., fragments that induce hematopoiesis). Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

FIGS. 3A-B show the nucleotide (SEQ ID NO:20) and deduced amino acid sequence (SEQ ID NO:21) corresponding to this gene.

FIG. 4 shows an analysis of the amino acid sequence (SEQ ID NO:21). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.

The data presented in FIG. 4 are also represented in tabular form in Table 10. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 4, and Table 10: “Res”: amino acid residue of SEQ ID NO:21 and FIGS. 3A-B; “Position”: position of the corresponding residue within SEQ ID NO:21 and FIGS. 3A-B; I: Alpha, Regions Garnier Robson; II: Alpha, Regions Chou Fasman; III: Beta, Regions Garnier Robson; IV: Beta, Regions Chou Fasman; V: Turn, Regions Garnier Robson; VI: Turn, Regions Chou Fasman; VII: Coil, Regions Garnier Robson; VIII: Hydrophilicity Plot Kyte Doolittle; IX: Hydrophobicity Plot Hopp Woods; X: Alpha, Amphipathic Regions Eisenberg; XI: Beta, Amphipathic Regions Eisenberg; XII: Flexible Regions Karplus Schulz; XIII: Antigenic Index Jameson Wolf; and XIV: Surface Probability Plot Emini.

Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consisting of, one or more of the following regions: alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in FIGS. 3A-B and/or Table 10, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 10 can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIGS. 3A-B, but may, as shown in Table 10, be represented or identified by using tabular representations of the data presented in FIG. 4 The DNA*STAR computer algorithm used to generate FIG. 4 set on the original default parameters) was used to present the data in FIG. 4 in a tabular format (See Table 10). The tabular format of the data in FIG. 4 is used to easily determine specific boundaries of a preferred region.

The present invention is further directed to fragments of the polynucleotide sequences described herein. By a fragment of, for example, the polynucleotide sequence of a deposited cDNA or the nucleotide sequence shown in SEQ ID NO:20, is intended polynucleotide fragments at least about 15 nt, and more preferably at least about 20 nt, at least about 25 nt, still more preferably at least about 30 nt, at least about 35 nt, and even more preferably, at least about 40 nt in length, at least about 45 nt in length, at least about 50 nt in length, at least about 60 nt in length, at least about 70 nt in length, at least about 80 nt in length, at least about 90 nt in length, at least about 100 nt in length, at least about 125 nt in length, at least about 150 nt in length, at least about 175 nt in length, which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 200-500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of a deposited cDNA or as shown in SEQ ID NO:20. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:20. In this context “about” includes the particularly recited size, an sizes larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, or from about 951 to about 985 of SEQ ID NO:20, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.

Preferred polypeptide fragments of the invention comprise, or alternatively consist of, the secreted protein having a continuous series of deleted residues from the amino or the carboxyl terminus, or both. Particularly, N-terminal deletions of the polypeptide can be described by the general formula m-136 where m is an integer from 2 to 136, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:21. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: R-2 to R-136; T-3 to R-136; P-4 to R-136; G-5 to R-136; P-6 to R-136; L-7 to R-136; P-8 to R-136; V-9 to R-136; L-10 to R-136; L-11 to R-136; L-12 to R-136; L-13 to R-136; L-14 to R-136; A-15 to R-136; G-16 to R-136; A-17 to R-136; P-18 to R-136; A-19 to R-136; A-20 to R-136; R-21 to R-136; P-22 to R-136; T-23 to R-136; P-24 to R-136; P-25 to R-136; T-26 to R-136; C-27 to R-136; Y-28 to R-136; S-29 to R-136; R-30 to R-136; M-31 to R-136; R-32 to R-136; A-33 to R-136; L-34 to R-136; S-35 to R-136; Q-36 to R-136; E-37 to R-136; 1-38 to R-136; T-39 to R-136; R-40 to R-136; D-41 to R-136; F-42 to R-136; N-43 to R-136; L-44 to R-136; L-45 to R-136; Q-46 to R-136; V-47 to R-136; S-48 to R-136; E-49 to R-136; P-50 to R-136; S-51 to R-136; E-52 to R-136; P-53 to R-136; C-54 to R-136; V-55 to R-136; R-56 to R-136; Y-57 to R-136; L-58 to R-136; P-59 to R-136; R-60 to R-136; L-61 to R-136; Y-62 to R-136; L-63 to R-136; D-64 to R-136; 1-65 to R-136; H-66 to R-136; N-67 to R-136; Y-68 to R-136; C-69 to R-136; V-70 to R-136; L-71 to R-136; D-72 to R-136; K-73 to R-136; L-74 to R-136; R-75 to R-136; D-76 to R-136; F-77 to R-136; V-78 to R-136; A-79 to R-136; S-80 to R-136; P-81 to R-136; P-82 to R-136; C-83 to R-136; W-84 to R-136; K-85 to R-136; V-86 to R-136; A-87 to R-136; Q-88 to R-136; V-89 to R-136; D-90 to R-136; S-91 to R-136; L-92 to R-136; K-93 to R-136; D-94 to R-136; K-95 to R-136; A-96 to R-136; R-97 to R-136; K-98 to R-136; L-99 to R-136; Y-100 to R-136; T-101 to R-136; 1-102 to R-136; M-103 to R-136; N-104 to R-136; S-105 to R-136; F-106 to R-136; C-107 to R-136; R-108 to R-136; R-109 to R-136; D-110 to R-136; L-111 to R-136; V-112 to R-136; F-113 to R-136; L-114 to R-136; L-115 to R-136; D-116 to R-136; D-117 to R-136; C-118 to R-136; N-119 to R-136; A-120 to R-136; L-121 to R-136; E-122 to R-136; Y-123 to R-136; P-124 to R-136; 1-125 to R-136; P-126 to R-136; V-127 to R-136; T-128 to R-136; T-129 to R-136; V-130 to R-136; and L-131 to R-136 of SEQ ID NO:21. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C terminus of a protein results in modification or loss of one or more biological functions of the protein (e.g., ability to induce hematopoiesis), other functional activities (e.g., biological activities, ability to multimerize, ability to bind receptors, ability to activate receptors, ability to bind and block receptor activation, ability to inhibit receptor activation without binding (e.g., as a dominant negative inhibitor of oligomeric complexes), ability to generate antibodies, ability to bind antibodies) may still be retained. For example the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C terminus. Whether a particular polypeptide lacking C terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a polypeptide with a large number of deleted C terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxyl terminus of the amino acid sequence of the polypeptide shown in FIGS. 3A-B (SEQ ID NO:21), as described by the general formula 1-n, where n is an integer from 6 to 135, where n corresponds to the position of the amino acid residue identified in SEQ ID NO:21. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to Q-135; M-1 to R-134; M-1 to D-133; M-1 to P-132; M-1 to L-131; M-1 to V-130; M-1 to T-129; M-1 to T-128; M-1 to V-127; M-1 to P-126; M-1 to I-125; M-1 to P-124; M-1 to Y-123; M-1 to E-122; M-1 to L-121; M-1 to A-120; M-1 to N-119; M-1 to C-118; M-1 to D-117; M-1 to D-116; M-1 to L-115; M-1 to L-114; M-1 to F-113; M-1 to V-112; M-1 to L-111; M-1 to D-110; M-1 to R-109; M-1 to R-108; M-1 to C-107; M-1 to F-106; M-1 to 5-105; M-1 to N-104; M-1 to M-103; M-1 to I-102; M-1 to T-101; M-1 to Y-100; M-1 to L-99; M-1 to K-98; M-1 to R-97; M-1 to A-96; M-1 to K-95; M-1 to D-94; M-1 to K-93; M-1 to L-92; M-1 to S-91; M-1 to D-90; M-1 to V-89; M-1 to Q-88; M-1 to A-87; M-1 to V-86; M-1 to K-85; M-1 to W-84; M-1 to C-83; M-1 to P-82; M-1 to P-81; M-1 to S-80; M-1 to A-79; M-1 to V-78; M-1 to F-77; M-1 to D-76; M-1 to R-75; M-1 to L-74; M-1 to K-73; M-1 to D-72; M-1 to L-71; M-1 to V-70; M-1 to C-69; M-1 to Y-68; M-1 to N-67; M-1 to H-66; M-1 to 1-65; M-1 to D-64; M-1 to L-63; M-1 to Y-62; M-1 to L-61; M-1 to R-60; M-1 to P-59; M-1 to L-58; M-1 to Y-57; M-1 to R-56; M-1 to V-55; M-1 to C-54; M-1 to P-53; M-1 to E-52; M-1 to 5-51; M-1 to P-50; M-1 to E-49; M-1 to S-48; M-1 to V-47; M-1 to Q-46; M-1 to L-45; M-1 to L-44; M-1 to N-43; M-1 to F-42; M-1 to D-41; M-1 to R-40; M-1 to T-39; M-1 to I-38; M-1 to E-37; M-1 to Q-36; M-1 to S-35; M-1 to L-34; M-1 to A-33; M-1 to R-32; M-1 to M-31; M-1 to R-30; M-1 to S-29; M-1 to Y-28; M-1 to C-27; M-1 to T-26; M-1 to P-25; M-1 to P-24; M-1 to T-23; M-1 to P-22; M-1 to R-21; M-1 to A-20; M-1 to A-19; M-1 to P-18; M-1 to A-17; M-1 to G-16; M-1 to A-15; M-1 to L-14; M-1 to L-13; M-1 to L-12; M-1 to L-11; M-1 to L-10; M-1 to V-9; M-1 to P-8; and M-1 to L-7 of SEQ ID NO:21. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In addition, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides comprising, or alternatively consisting of, one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of SEQ ID NO:21, where n and m are integers as described above. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to A-15; R-2 to G-16; T-3 to A-17; P-4 to P-18; G-5 to A-19; P-6 to A-20; L-7 to R-21; P-8 to P-22; V-9 to T-23; L-10 to P-24; L-11 to P-25; L-12 to T-26; L-13 to C-27; L-14 to Y-28; A-15 to S-29; G-16 to R-30; A-17 to M-31; P-18 to R-32; A-19 to A-33; A-20 to L-34; R-21 to S-35; P-22 to Q-36; T-23 to E-37; P-24 to I-38; P-25 to T-39; T-26 to R-40; C-27 to D-41; Y-28 to F-42; S-29 to N-43; R-30 to L-44; M-31 to L-45; R-32 to Q-46; A-33 to V-47; L-34 to S-48; S-35 to E-49; Q-36 to P-50; E-37 to S-51; 1-38 to E-52; T-39 to P-53; R-40 to C-54; D-41 to V-55; F-42 to R-56; N-43 to Y-57; L-44 to L-58; L-45 to P-59; Q-46 to R-60; V-47 to L-61; S-48 to Y-62; E-49 to L-63; P-50 to D-64; S-51 to I-65; E-52 to H-66; P-53 to N-67; C-54 to Y-68; V-55 to C-69; R-56 to V-70; Y-57 to L-71; L-58 to D-72; P-59 to K-73; R-60 to L-74; L-61 to R-75; Y-62 to D-76; L-63 to F-77; D-64 to V-78; 1-65 to A-79; H-66 to S-80; N-67 to P-81; Y-68 to P-82; C-69 to C-83; V-70 to W-84; L-71 to K-85; D-72 to V-86; K-73 to A-87; L-74 to Q-88; R-75 to V-89; D-76 to D-90; F-77 to S-91; V-78 to L-92; A-79 to K-93; S-80 to D-94; P-81 to K-95; P-82 to A-96; C-83 to R-97; W-84 to K-98; K-85 to L-99; V-86 to Y-100; A-87 to T-101; Q-88 to I-102; V-89 to M-103; D-90 to N-104; S-91 to S-105; L-92 to F-106; K-93 to C-107; D-94 to R-108; K-95 to R-109; A-96 to D-110; R-97 to L-111; K-98 to V-112; L-99 to F-113; Y-100 to L-114; T-101 to L-115; I-102 to D-116; M-103 to D-117; N-104 to C-118; S-105 to N-119; F-106 to A-120; C-107 to L-121; R-108 to E-122; R-109 to Y-123; D-110 to P-124; L-111 to I-125; V-112 to P-126; F-113 to V-127; L-114 to T-128; L-115 to T-129; D-116 to V-130; D-117 to L-131; C-118 to P-132; N-119 to D-133; A-120 to R-134; L-121 to Q-135; and E-122 to R-136 of SEQ ID NO:21. Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The present invention is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein as m-n. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N and C terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also included are polynucleotide sequences encoding a polypeptide consisting of a portion of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 97975 (deposited Apr. 4, 1997) and ATCC Deposit No. 209081 (deposited May 29, 1997), where this portion excludes any integer of amino acid residues from 1 to about 606 (end of protein minus six) amino acids from the amino terminus of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 97975 and 209081, or any integer of amino acid residues from 6 to about 612 amino acids from the carboxyl terminus, or any combination of the above amino terminal and carboxyl terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97975 and 209081. Polypeptides encoded by these polynucleotides also are encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The gene encoding the disclosed cDNA is believed to reside on chromosome 4. Accordingly, polynucleotides related to this invention have uses that include, but are not limited to, serving as probes or primers in chromosome identification, chromosome mapping, and linkage analysis for chromosome 4.

This gene is expressed primarily in fetal liver and fetal spleen.

Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, hematopoietic, immunological, developmental, and/or hepatic disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and hematopoetic systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., hematopoietic, immune, hepatic, developmental, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, bile, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. For example, polynucleotides and polypeptides of the invention, polynucleotide and polypeptide fragments, and polynucleotide and polypeptide variants, and antibodies directed to these polypeptides are useful for identifying, selecting, targeting and/or stimulating proliferation of hematopoietic stem cells (a.k.a., hematopoietic progenitor cells).

Cytokines typically exert their respective biochemical and physiological effects by binding to specific receptor molecules. Receptor binding then stimulates specific signal transduction pathways (Kishimoto, T., et al., Cell 76:253 262 (1994)). The specific interactions of cytokines with their receptors are often the primary regulators of a wide variety of cellular processes including activation, proliferation, and differentiation (Arai, K. I, et al., Ann. Rev. Biochem. 59:783 836 (1990); Paul, W. E. and Seder, R. A., Cell 76:241 251 (1994)).

The polynucleotides and polypeptides of this invention may be useful for the diagnosis and treatment of a variety of immune system and hematopoietic disorders, pathologies, and/or deficiencies. For example, this gene and/or gene product may play a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. Furthermore, polypeptides of this invention may be involved in the regulation of cytokine production, antigen presentation, or other processes useful for treatment of cancer, particularly leukemia (e.g., by boosting immune responses, suppressing hyperproliferative activity, or enhancing recovery of healthy hematopoietic cell populations during or following chemotherapy). Moreover, the polynucleotides and polypeptides of this invention, as well as antibodies against the polypeptides of this invention, may be useful for treating immunological and hematopoietic disorders; such as for examples, arthritis, asthma, immunodeficiency diseases (e.g. AIDS), leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma. Moreover, the polypeptide of this invention represents a secreted factor that is likely to have activity in stimulating the differentiation of blood cells, or recruiting immune and hematopoietic cells to sites of injury. Thus, this polypeptide is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

Preferred polypeptides of the present invention comprise, or alternatively consist of, one or more of the immunogenic epitopes shown in SEQ ID NO:21 as residues: Met-1 to Leu-7, Pro-18 to Cys-27, Ser-29 to Ser-35, Glu-37 to Asp-41, Gln-46 to Cys-54, Asp-72 to Val-78, Pro-81 to Trp-84, Ser-91 to Lys-98, Asn-104 to Leu-111, Asp-116 to Leu-121, and Val-130 to Arg-136. Polynucleotides encoding said polypeptides are also encompassed by the invention. Antibodies that bind said epitopes or other polypeptides of the invention are also encompassed.

The tissue distribution of this gene in fetal liver and spleen indicates that the gene could be important for the treatment or detection of immune or hematopoietic disorders including arthritis, leukemia, and immunodeficiency diseases. Moreover, the protein product of this gene is useful for the treatment and diagnosis of hematopoetic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia. The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Moreover, expression within fetal tissue indicates that this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus, this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:20 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 982 of SEQ ID NO:20, b is an integer of 15 to 996, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:20, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 6

The translation product of this gene is homologous to the Clostridium perfringens enterotoxin (CPE) receptor gene product and shares sequence homology with a human ORF specific to prostate and a glycoprotein specific to oligodendrocytes, both of which are tissue specific proteins. See e.g., Katahira et al. J. Cell Biol. 136(6):1239-1247 (1997). PMID: 9087440; UI: 97242441.

In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequences: TMASMGLQV (SEQ ID NO:30), KSWMMLWAVQDTGTITIRPANRNTTPATIMVLALALSSSRQLVHLPPTTDSSTPRAATMM LMMTRARAACRSCGSASSESYTLHClWPVLCTTQFIHRPSQMVCEVTMLLPMKAVTRHMG SAQHSMTASQPRTASAMPITCSPMEAIVQRPRELRTWKAEGIRLWGP (SEQ ID NO:31), LQVMGIALAVLGWLAVMLCCALPMWRVT (SEQ ID NO:32), SNIVTSQTIWEGLWMNCVVQST (SEQ ID NO:33), QMQCKVYDSLLALPQDLQ (SEQ ID NO:34), KCTNCLEDESAKAKTMIV (SEQ ID NO:35), GVVFLLAGLMVIVPVSWTAHNIIQDFYNPLVA (SEQ ID NO:36), and/or CCNCPPRTDKPY (SEQ ID NO:37). Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The gene encoding the disclosed cDNA is believed to reside on chromosome 7. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 7.

This gene is expressed primarily in pancreas tumor and ulcerative colitis, and to a lesser extent in several tumors and normal tissues.

Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, metabolic, gastrointestinal, or proliferative disorders, such as pancreatic disorders, ulcerative colitis, tumors and food poisoning. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the digestive system or tumorigenic system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., metabolic, gastrointestinal, pancreatic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, bile, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Predicted epitopes include those comprising a sequence shown in SEQ ID NO:29 as residues: Gly-147 to Met-152, Cys-177 to Lys-188.

The tissue distribution in pancreas, combined with the homology to a prostate and oligodendrocyte-specific protein, indicates that the protein product of this gene is useful as a marker for the diagnosis or treatment of disorders in pancreas, ulcerative colitis, and tumors. Furthermore, identity to the human receptor for Clostridium perfringenes enterotoxin indicates that the soluble portion of this receptor could be used in the treatment of food poisoning associated with Clostridia perfringens by blocking the activity of the perfringens enterotoxin. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:28 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1691 of SEQ ID NO:28, b is an integer of 15 to 1705, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:28, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 7

The translation product of this gene shares sequence homology with alpha 3 type IX collagen, which is thought to be important in hyaline cartilage formation via its ability to uptake inorganic sulfate by cells (See Genbank Accession No. gi|975657).

In specific embodiments, polypeptides of the invention comprise, or alternatively consists of, an amino acid sequence selected from the group: SLRRPRSAAXQTLTTFLSSVSSASSSALPGSREPCDPRAPPPPRSGSAASCCSCCCSCPRRRAP LRSPRGSKRRIRQREVVDLYNGMCLQGPAGVPGRDGSPGANGIPGTPGIPGRDGFKGEKGE CLRESFEESWTPNYKQCSWSSLNYGIDLGKIAECTFTKMRSNSALRVLFSGSLRLKCRNAC CQRWYFTFNGAECSGPLPIEAIIYLDQGSPEMNSTINIHRTSSVEGLCEGIGAGLVDVAIWVG TCSDYPKGDASTGWNSVSRIIIEELPK (SEQ ID NO:40), SLRRPRSAAXQTLTTFLSSVSSASSSALPGSREPCDPRAPPPPRSGSAASCCSCCCSCPRR (SEQ ID NO:41), RAPLRSPRGSKRRIRQREVVDLYNGMCLQGPAGVPGRDGSPGANGIPGTPGI (SEQ ID NO:42), TPGIPGRDGFKGEKGECLRESFEESWTPNYKQCSWSSLNYGIDLGKIAECTF (SEQ ID NO:43), FTKMRSNSALRVLFSGSLRLKCRNACCQRWYFTFNGAECSGPLPIEAIIYLDQGSPEMNSTI NIHR (SEQ ID NO:44), and/or RTSSVEGLCEGIGAGLVDVAIWVGTCSDYPKGDASTGWNSVSRIIIEELPK (SEQ ID NO:45). Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in smooth muscle, and to a lesser extent in synovial tissue.

Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias, i.e. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid and autoimmune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the skeletal system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g. muscle, synovial tissues, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in smooth muscle, and homology to alpha 3 type IX collagen indicates that the protein product of this gene is useful for the treatment and diagnosis of diseases associated with the mutation in this gene which leads to the many different types of chondrodysplasias. By the use of this product, the abnormal growth and development of bones of the limbs and spine could be detected or treated in utero, since the protein or polypeptides thereof could affect epithelial cells early in development, and later the chondrocytes of the developing craniofacial structure. In addition, the expression of this gene product in synovium would suggest a role in the detection and treatment of disorders and conditions affecting the skeletal system, in particular osteoporosis as well as disorders afflicting connective tissues (e.g. arthritis, trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis or treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (i.e. spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). Moreover, the expression within smooth muscle indicates t that polynucleotides and polypeptides corresponding to this gene are useful for the treatment, detection, and/or prevention of a variety of vascular disorders, which include, but are not limited to, atherosclerosis, embolism, stroke, aneurysm, or microvascular disease. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:38 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1274 of SEQ ID NO:38, b is an integer of 15 to 1288, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:38, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 8

The translation product of this gene shares sequence homology with both the RIC and MAT8 proteins (mouse), which are thought to be important in regulating chloride conductance in cells by modulating the response mediated by cAMP and protein kinase C to extracellular signals.

In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequences: GTSLDAAATAASLSPRGCRLRTPSSD (SEQ ID NO:52), QIQRHTRAPKQLIPLMTPRRSLRDHPQAQTSRQTPRPSSHLVFMRMTPSSMMNTPSGNGGC WSQLCCSSQASSSSPVASAGSCPGYAGIIAGESIRNRS (SEQ ID NO:53), PRRSLRDHPQAQTSRQTPRPSSHLVFM (SEQ ID NO:54), THPPETGAVGRSCAVHHRHHHPHQWQVQAAVPVMPESLQVSPSETGADNXLGTRRPSPLP AHRAQPPASPRRAWPEREDTDDEAGARAAGPSLLPPPTLPAPEGYLAPWGLSLKLSPLLRQ KVKHCGLC (SEQ ID NO:55), PESLQVSPSETGADNXLGTRRPSPLPAHRAQPPASP (SEQ ID NO:56), GTAPKAPGSLQGRAGLGEVGDSDRQPWLQLHHLCLPSLARLFEGMQEAGHGELAGGLVF GCPAGCQLLFLMDSPAMIPA (SEQ ID NO:57), GEVGDSDRQPWLQLHHLCLPSLARLFEGMQEAGH (SEQ ID NO:58), GSGGLSGRLCLGMVSQRASWCHQWDELLWCSCVSLDLSLEAHPFLPVAGSGSGVVVFHQ QARLGLERWAGVLCRLHLGLVSGPECP (SEQ ID NO:59), and/or QWDELLWCSCVSLDLSLEAHPFLPVAGSGSGVVVFHQQARL (SEQ ID NO:60). Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The gene encoding the disclosed cDNA is believed to reside on chromosome 19. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 19.

This gene is expressed primarily in amniotic cells and hematopoietic cells including macrophages, neutrophils, T cells, TNF induced aortic endothelium, and to a lesser extent in testes, TNF induced epithelial cells, and smooth muscle.

Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune or hematopoietic disorders, particularly inflammatory responses mediated by T cells, macrophages, and/or neutrophils, particularly those involving TNF, and also cancer. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, developmental, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Predicted epitopes include those comprising a sequence shown in SEQ ID NO:47 as residues: Thr-19 to Ala-33, Leu-54 to Asp-82, Pro-89 to Ala-97, Pro-100 to Lys-125, Ser-127 to Phe-135, Gly-164 to Leu-169, Cys-173 to Arg-178.

The tissue distribution in hematopoietic cells, combined with the homology to the RIC and mat-8 genes, indicates that the protein product of this gene is useful for modifying inflammatory responses to cytokines such as TNF, and thus modifying the duration and/or severity of inflammation. Polynucleotides and polypeptides derived from this gene are thought to be useful in the diagnosis and treatment of cancer. The protein product of this gene is useful for the treatment and diagnosis of hematopoetic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia, since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia. The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:46 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 927 of SEQ ID NO:46, b is an integer of 15 to 941, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:46, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 9

In specific embodiments, polypeptides of the invention comprise, or alternatively consists of, an amino acid sequence selected from the group: MRILQLILLALATGLVGGETRIIKGFECKLHSQPWQAALFEKTRLLCGATLIAPRWLLTAAH CLKPRYIVHLGQHNLQKEEGCEQTRTATESFPHPGFNNSLPNKDHRNDIMLVKMASPVSIT WAVRPLTLSSRCVTAGTSCSFPAGAARPDPSYACLTPCDAPTSPSLSTRSVRTPTPATSQTP WCVPACRKGARTPARVTPGALWSVTSLFKALSPGARIRVRSPESLVSTRKSANMWTGSRR R (SEQ ID NO:67); ETRIIKGFECKLHSQPWQAALFEKTRLLCGATLIAPRWLLTAAHCLKPRYIVHLGQHNLQK EEGCEQTRTATESFPHPGFNNSLPNKDHRNDIMLVKMASPVSITWAVRPLTLSSRCVTAGT SCSFPAGAARPDPSYACLTPCDAPTSPSLSTRSVRTPTPATSQTPWCVPACRKGARTPARVT PGALWSVTSLFKALSPGARIRVRSPESLVSTRKSANMWTGSRRR (SEQ ID NO:68); and/or CKLHSQPWQAALFEKTRLLCGATLIAPRWLLTAAHCLKPRYIVHLGQHNLQKEEGCEQTR TATESFPHPGFNNS (SEQ ID NO:69). Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The translation product of this gene shares sequence homology with neuropsin, a novel serine protease, which is thought to be important in modulating extracellular signaling pathways in the brain. Owing to the structural similarity to other serine proteases, the protein products of this gene are expected to have serine protease activity which may be assayed by methods known in the art and described elsewhere herein. Moreover, this protein has been shown to also have homology to PSA (prostate specific antigen). PSA is a serum marker for prostate cancer and it is a member of the kallikrein family. The members of the kallikrein family are secreted serine proteases and some of them are good tissue specific markers. This new member of the kallikrein family has been detected twice in endometrial tumor cDNA library and therefore is a good candidate as a serum marker for endometrial tumor.

This gene is expressed primarily in endometrial tumor, and to a lesser extent, in colon cancer, benign hypertrophic prostate, and thymus.

Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, reproductive, immune, or endocrine disorders, particularly cancers of the endometrium or colon and benign hypertrophy of the prostate. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the urogenital or reproductive systems, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, immune, endocrine, gastrointestinal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, seminal fluid, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Predicted epitopes include those comprising a sequence shown in SEQ ID NO:62 as residues: Glu-27 to Trp-35, Leu-77 to Ala-89, Pro-96 to Asn-109, Ser-149 to Arg-156, Gln-172 to Ile-182, Glu-193 to Gly-204, Glu-245 to Asn-250.

The tissue distribution in proliferative reproductive tissues, combined with the homology to serine proteases indicates that the protein product of this gene is useful for diagnosing, treating, and/or preventing hyperproliferative disorders such as cancer of the endometrium or colon and hyperplasia of the prostate. Similarly, expression within cellular sources marked by proliferating cells indicates that this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:61 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1278 of SEQ ID NO:61, b is an integer of 15 to 1292, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:61, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 10

The translation product of this gene shares sequence homology with tenascin, which is thought to be important in development. The translation product of this gene is believed to be a ligand of the fibroblast growth factor family. FGF ligand activity is known in the art and can be assayed by methods known in the art and disclosed elsewhere herein.

Northern analysis indicates that a 2.5 kb band is expressed in brain and lung. It has also been discovered that this gene is expressed in endometrial tumor, synovial sarcoma, pancreas tumor, fetal lung, retinal, and immune tissues (e.g., bone marrow)

Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, cancers, growth disorders of the brain and lung. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the cancer tissues, brain, lung, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g. brain, lung, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Predicted epitopes include those comprising a sequence shown in SEQ ID NO:71 as residues: Gly-29 to Glu-34, Arg-71 to Arg-76, Thr-176 to Cys-182, Gly-184 to Glu-199. As a preferred embodiment, antibodies that bind said epitopes are encompassed by the invention and may be useful as a cancer diagnostic and/or an agonist/antagonist of the polypeptides of the invention.

Fragments and variants of the polypeptide encoded by this gene (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention). Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention. Antibodies that bind polypeptides of the invention would be useful as a cancer diagnostic.

Preferred polypeptide fragments of the invention comprise, or alternatively consist of, the secreted protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. Particularly, N-terminal deletions of the polypeptide can be described by the general formula m-379 where m is an integer from 2 to 371, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:71. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: A-2 to W-379; R-3 to W-379; R-4 to W-379; S-5 to W-379; A-6 to W-379; F-7 to W-379; P-8 to W-379; A-9 to W-379; A-10 to W-379; A-11 to W-379; L-12 to W-379; W-13 to W-379; L-14 to W-379; W-15 to W-379; S-16 to W-379; 1-17 to W-379; L-18 to W-379; L-19 to W-379; C-20 to W-379; L-21 to W-379; L-22 to W-379; A-23 to W-379; L-24 to W-379; R-25 to W-379; A-26 to W-379; E-27 to W-379; A-28 to W-379; G-29 to W-379; P-30 to W-379; P-31 to W-379; Q-32 to W-379; E-33 to W-379; E-34 to W-379; S-35 to W-379; L-36 to W-379; Y-37 to W-379; L-38 to W-379; W-39 to W-379; 1-40 to W-379; D-41 to W-379; A-42 to W-379; H-43 to W-379; Q-44 to W-379; A-45 to W-379; R-46 to W-379; V-47 to W-379; L-48 to W-379; 1-49 to W-379; G-50 to W-379; F-51 to W-379; E-52 to W-379; E-53 to W-379; D-54 to W-379; I-55 to W-379; L-56 to W-379; 1-57 to W-379; V-58 to W-379; 5-59 to W-379; E-60 to W-379; G-61 to W-379; K-62 to W-379; M-63 to W-379; A-64 to W-379; P-65 to W-379; F-66 to W-379; T-67 to W-379; H-68 to W-379; D-69 to W-379; F-70 to W-379; R-71 to W-379; K-72 to W-379; A-73 to W-379; Q-74 to W-379; Q-75 to W-379; R-76 to W-379; M-77 to W-379; P-78 to W-379; A-79 to W-379; 1-80 to W-379; P-81 to W-379; V-82 to W-379; N-83 to W-379; I-84 to W-379; H-85 to W-379; 5-86 to W-379; M-87 to W-379; N-88 to W-379; F-89 to W-379; T-90 to W-379; W-91 to W-379; Q-92 to W-379; A-93 to W-379; A-94 to W-379; G-95 to W-379; Q-96 to W-379; A-97 to W-379; E-98 to W-379; Y-99 to W-379; F-100 to W-379; Y-101 to W-379; E-102 to W-379; F-103 to W-379; L-104 to W-379; S-105 to W-379; L-106 to W-379; R-107 to W-379; S-108 to W-379; L-109 to W-379; D-110 to W-379; K-111 to W-379; G-112 to W-379; 1-113 to W-379; M-114 to W-379; A-115 to W-379; D-116 to W-379; P-117 to W-379; T-118 to W-379; V-119 to W-379; N-120 to W-379; V-121 to W-379; P-122 to W-379; L-123 to W-379; L-124 to W-379; G-125 to W-379; T-126 to W-379; V-127 to W-379; P-128 to W-379; H-129 to W-379; K-130 to W-379; A-131 to W-379; S-132 to W-379; V-133 to W-379; V-134 to W-379; Q-135 to W-379; V-136 to W-379; G-137 to W-379; F-138 to W-379; P-139 to W-379; C-140 to W-379; L-141 to W-379; G-142 to W-379; K-143 to W-379; Q-144 to W-379; D-145 to W-379; G-146 to W-379; V-147 to W-379; A-148 to W-379; A-149 to W-379; F-150 to W-379; E-151 to W-379; V-152 to W-379; D-153 to W-379; V-154 to W-379; 1-155 to W-379; V-156 to W-379; M-157 to W-379; N-158 to W-379; S-159 to W-379; E-160 to W-379; G-161 to W-379; N-162 to W-379; T-163 to W-379; 1-164 to W-379; L-165 to W-379; Q-166 to W-379; T-167 to W-379; P-168 to W-379; Q-169 to W-379; N-170 to W-379; A-171 to W-379; I-172 to W-379; F-173 to W-379; F-174 to W-379; K-175 to W-379; T-176 to W-379; C-177 to W-379; Q-178 to W-379; Q-179 to W-379; A-180 to W-379; E-181 to W-379; C-182 to W-379; P-183 to W-379; G-184 to W-379; G-185 to W-379; C-186 to W-379; R-187 to W-379; N-188 to W-379; G-189 to W-379; G-190 to W-379; F-191 to W-379; C-192 to W-379; N-193 to W-379; E-194 to W-379; R-195 to W-379; R-196 to W-379; 1-197 to W-379; C-198 to W-379; E-199 to W-379; C-200 to W-379; P-201 to W-379; D-202 to W-379; G-203 to W-379; F-204 to W-379; H-205 to W-379; G-206 to W-379; P-207 to W-379; H-208 to W-379; C-209 to W-379; E-210 to W-379; K-211 to W-379; A-212 to W-379; L-213 to W-379; C-214 to W-379; T-215 to W-379; P-216 to W-379; R-217 to W-379; C-218 to W-379; M-219 to W-379; N-220 to W-379; G-221 to W-379; G-222 to W-379; L-223 to W-379; C-224 to W-379; V-225 to W-379; T-226 to W-379; P-227 to W-379; G-228 to W-379; F-229 to W-379; C-230 to W-379; 1-231 to W-379; C-232 to W-379; P-233 to W-379; P-234 to W-379; G-235 to W-379; F-236 to W-379; Y-237 to W-379; G-238 to W-379; V-239 to W-379; N-240 to W-379; C-241 to W-379; D-242 to W-379; K-243 to W-379; A-244 to W-379; N-245 to W-379; C-246 to W-379; S-247 to W-379; T-248 to W-379; T-249 to W-379; C-250 to W-379; F-251 to W-379; N-252 to W-379; G-253 to W-379; G-254 to W-379; T-255 to W-379; C-256 to W-379; F-257 to W-379; Y-258 to W-379; P-259 to W-379; G-260 to W-379; K-261 to W-379; C-262 to W-379; I-263 to W-379; C-264 to W-379; P-265 to W-379; P-266 to W-379; G-267 to W-379; L-268 to W-379; E-269 to W-379; G-270 to W-379; E-271 to W-379; Q-272 to W-379; C-273 to W-379; E-274 to W-379; 1-275 to W-379; S-276 to W-379; K-277 to W-379; C-278 to W-379; P-279 to W-379; Q-280 to W-379; P-281 to W-379; C-282 to W-379; R-283 to W-379; N-284 to W-379; G-285 to W-379; G-286 to W-379; K-287 to W-379; C-288 to W-379; 1-289 to W-379; G-290 to W-379; K-291 to W-379; S-292 to W-379; K-293 to W-379; C-294 to W-379; K-295 to W-379; C-296 to W-379; S-297 to W-379; K-298 to W-379; G-299 to W-379; Y-300 to W-379; Q-301 to W-379; G-302 to W-379; D-303 to W-379; L-304 to W-379; C-305 to W-379; S-306 to W-379; K-307 to W-379; P-308 to W-379; V-309 to W-379; C-310 to W-379; E-311 to W-379; P-312 to W-379; G-313 to W-379; C-314 to W-379; G-315 to W-379; A-316 to W-379; H-317 to W-379; G-318 to W-379; T-319 to W-379; C-320 to W-379; H-321 to W-379; E-322 to W-379; P-323 to W-379; N-324 to W-379; K-325 to W-379; C-326 to W-379; Q-327 to W-379; C-328 to W-379; Q-329 to W-379; E-330 to W-379; G-331 to W-379; W-332 to W-379; H-333 to W-379; G-334 to W-379; R-335 to W-379; H-336 to W-379; C-337 to W-379; N-338 to W-379; K-339 to W-379; R-340 to W-379; Y-341 to W-379; E-342 to W-379; A-343 to W-379; S-344 to W-379; L-345 to W-379; 1-346 to W-379; H-347 to W-379; A-348 to W-379; L-349 to W-379; R-350 to W-379; P-351 to W-379; A-352 to W-379; G-353 to W-379; A-354 to W-379; Q-355 to W-379; L-356 to W-379; R-357 to W-379; Q-358 to W-379; H-359 to W-379; T-360 to W-379; P-361 to W-379; S-362 to W-379; L-363 to W-379; K-364 to W-379; K-365 to W-379; A-366 to W-379; E-367 to W-379; E-368 to W-379; R-369 to W-379; R-370 to W-379; D-371 to W-379; P-372 to W-379; P-373 to W-379; and E-374 to W-379 of SEQ ID NO:71. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind ligand, ability to generate antibodies, ability to bind antibodies) may still be retained. For example the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C terminus. Whether a particular polypeptide lacking C terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a polypeptide with a large number of deleted C terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in SEQ ID NO:71, as described by the general formula 1-n, where n is an integer from 6 to 378, where n corresponds to the position of the amino acid residue identified in SEQ ID NO:71. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to I-378; M-1 to Y-377; M-1 to N-376; M-1 to S-375; M-1 to E-374; M-1 to P-373; M-1 to P-372; M-1 to D-371; M-1 to R-370; M-1 to R-369; M-1 to E-368; M-1 to E-367; M-1 to A-366; M-1 to K-365; M-1 to K-364; M-1 to L-363; M-1 to S-362; M-1 to P-361; M-1 to T-360; M-1 to H-359; M-1 to Q-358; M-1 to R-357; M-1 to L-356; M-1 to Q-355; M-1 to A-354; M-1 to G-353; M-1 to A-352; M-1 to P-351; M-1 to R-350; M-1 to L-349; M-1 to A-348; M-1 to H-347; M-1 to I-346; M-1 to L-345; M-1 to S-344; M-1 to A-343; M-1 to E-342; M-1 to Y-341; M-1 to R-340; M-1 to K-339; M-1 to N-338; M-1 to C-337; M-1 to H-336; M-1 to R-335; M-1 to G-334; M-1 to H-333; M-1 to W-332; M-1 to G-331; M-1 to E-330; M-1 to Q-329; M-1 to C-328; M-1 to Q-327; M-1 to C-326; M-1 to K-325; M-1 to N-324; M-1 to P-323; M-1 to E-322; M-1 to H-321; M-1 to C-320; M-1 to T-319; M-1 to G-318; M-1 to H-317; M-1 to A-316; M-1 to G-315; M-1 to C-314; M-1 to G-313; M-1 to P-312; M-1 to E-311; M-1 to C-310; M-1 to V-309; M-1 to P-308; M-1 to K-307; M-1 to S-306; M-1 to C-305; M-1 to L-304; M-1 to D-303; M-1 to G-302; M-1 to Q-301; M-1 to Y-300; M-1 to G-299; M-1 to K-298; M-1 to S-297; M-1 to C-296; M-1 to K-295; M-1 to C-294; M-1 to K-293; M-1 to S-292; M-1 to K-291; M-1 to G-290; M-1 to I-289; M-1 to C-288; M-1 to K-287; M-1 to G-286; M-1 to G-285; M-1 to N-284; M-1 to R-283; M-1 to C-282; M-1 to P-281; M-1 to Q-280; M-1 to P-279; M-1 to C-278; M-1 to K-277; M-1 to S-276; M-1 to I-275; M-1 to E-274; M-1 to C-273; M-1 to Q-272; M-1 to E-271; M-1 to G-270; M-1 to E-269; M-1 to L-268; M-1 to G-267; M-1 to P-266; M-1 to P-265; M-1 to C-264; M-1 to I-263; M-1 to C-262; M-1 to K-261; M-1 to G-260; M-1 to P-259; M-1 to Y-258; M-1 to F-257; M-1 to C-256; M-1 to T-255; M-1 to G-254; M-1 to G-253; M-1 to N-252; M-1 to F-251; M-1 to C-250; M-1 to T-249; M-1 to T-248; M-1 to S-247; M-1 to C-246; M-1 to N-245; M-1 to A-244; M-1 to K-243; M-1 to D-242; M-1 to C-241; M-1 to N-240; M-1 to V-239; M-1 to G-238; M-1 to Y-237; M-1 to F-236; M-1 to G-235; M-1 to P-234; M-1 to P-233; M-1 to C-232; M-1 to I-231; M-1 to C-230; M-1 to F-229; M-1 to G-228; M-1 to P-227; M-1 to T-226; M-1 to V-225; M-1 to C-224; M-1 to L-223; M-1 to G-222; M-1 to G-221; M-1 to N-220; M-1 to M-219; M-1 to C-218; M-1 to R-217; M-1 to P-216; M-1 to T-215; M-1 to C-214; M-1 to L-213; M-1 to A-212; M-1 to K-211; M-1 to E-210; M-1 to C-209; M-1 to H-208; M-1 to P-207; M-1 to G-206; M-1 to H-205; M-1 to F-204; M-1 to G-203; M-1 to D-202; M-1 to P-201; M-1 to C-200; M-1 to E-199; M-1 to C-198; M-1 to I-197; M-1 to R-196; M-1 to R-195; M-1 to E-194; M-1 to N-193; M-1 to C-192; M-1 to F-191; M-1 to G-190; M-1 to G-189; M-1 to N-188; M-1 to R-187; M-1 to C-186; M-1 to G-185; M-1 to G-184; M-1 to P-183; M-1 to C-182; M-1 to E-181; M-1 to A-180; M-1 to Q-179; M-1 to Q-178; M-1 to C-177; M-1 to T-176; M-1 to K-175; M-1 to F-174; M-1 to F-173; M-1 to I-172; M-1 to A-171; M-1 to N-170; M-1 to Q-169; M-1 to P-168; M-1 to T-167; M-1 to Q-166; M-1 to L-165; M-1 to I-164; M-1 to T-163; M-1 to N-162; M-1 to G-161; M-1 to E-160; M-1 to S-159; M-1 to N-158; M-1 to M-157; M-1 to V-156; M-1 to I-155; M-1 to V-154; M-1 to D-153; M-1 to V-152; M-1 to E-151; M-1 to F-150; M-1 to A-149; M-1 to A-148; M-1 to V-147; M-1 to G-146; M-1 to D-145; M-1 to Q-144; M-1 to K-143; M-1 to G-142; M-1 to L-141; M-1 to C-140; M-1 to P-139; M-1 to F-138; M-1 to G-137; M-1 to V-136; M-1 to Q-135; M-1 to V-134; M-1 to V-133; M-1 to S-132; M-1 to A-131; M-1 to K-130; M-1 to H-129; M-1 to P-128; M-1 to V-127; M-1 to T-126; M-1 to G-125; M-1 to L-124; M-1 to L-123; M-1 to P-122; M-1 to V-121; M-1 to N-120; M-1 to V-119; M-1 to T-118; M-1 to P-117; M-1 to D-116; M-1 to A-115; M-1 to M-114; M-1 to I-113; M-1 to G-112; M-1 to K-111; M-1 to D-110; M-1 to L-109; M-1 to S-108; M-1 to R-107; M-1 to L-106; M-1 to S-105; M-1 to L-104; M-1 to F-103; M-1 to E-102; M-1 to Y-101; M-1 to F-100; M-1 to Y-99; M-1 to E-98; M-1 to A-97; M-1 to Q-96; M-1 to G-95; M-1 to A-94; M-1 to A-93; M-1 to Q-92; M-1 to W-91; M-1 to T-90; M-1 to F-89; M-1 to N-88; M-1 to M-87; M-1 to S-86; M-1 to H-85; M-1 to I-84; M-1 to N-83; M-1 to V-82; M-1 to P-81; M-1 to I-80; M-1 to A-79; M-1 to P-78; M-1 to M-77; M-1 to R-76; M-1 to Q-75; M-1 to Q-74; M-1 to A-73; M-1 to K-72; M-1 to R-71; M-1 to F-70; M-1 to D-69; M-1 to H-68; M-1 to T-67; M-1 to F-66; M-1 to P-65; M-1 to A-64; M-1 to M-63; M-1 to K-62; M-1 to G-61; M-1 to E-60; M-1 to S-59; M-1 to V-58; M-1 to I-57; M-1 to L-56; M-1 to I-55; M-1 to D-54; M-1 to E-53; M-1 to E-52; M-1 to F-51; M-1 to G-50; M-1 to I-49; M-1 to L-48; M-1 to V-47; M-1 to R-46; M-1 to A-45; M-1 to Q-44; M-1 to H-43; M-1 to A-42; M-1 to D-41; M-1 to I-40; M-1 to W-39; M-1 to L-38; M-1 to Y-37; M-1 to L-36; M-1 to S-35; M-1 to E-34; M-1 to E-33; M-1 to Q-32; M-1 to P-31; M-1 to P-30; M-1 to G-29; M-1 to A-28; M-1 to E-27; M-1 to A-26; M-1 to R-25; M-1 to L-24; M-1 to A-23; M-1 to L-22; M-1 to L-21; M-1 to C-20; M-1 to L-19; M-1 to L-18; M-1 to I-17; M-1 to 5-16; M-1 to W-15; M-1 to L-14; M-1 to W-13; M-1 to L-12; M-1 to A-11; M-1 to A-10; M-1 to A-9; M-1 to P-8; M-1 to F-7; and M-1 to A-6 of SEQ ID NO:71. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The tissue distribution in brain and lung, combined with the homology to tenascin indicates that the protein product of this gene is useful for diagnosis and treatment of cancers. Alternatively, given the tissue distribution indicated by Northern analysis, the translation product of this gene is thought to be a growth factor functioning in the brain and lung that may be useful in treating neurodegeneration and lung disorder. For example, the protein product of this gene is useful for the detection and treatment of disorders associated with developing lungs, particularly in premature infants where the lungs are the last tissues to develop. Furthermore, the tissue distribution indicates that the protein product of this gene is useful for the diagnosis and intervention of lung tumors, since the gene may be involved in the regulation of cell division. Additionally, expression in the brain indicates that it may be involved in neuronal survival; synapse formation; conductance; neural differentiation, etc. Such involvement may impact many processes, such as learning and cognition. It may also be useful in the treatment of such neurodegenerative disorders as schizophrenia; ALS; or Alzheimer's.

Polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51, and elsewhere herein. Briefly, the uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia. The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

Protein, as well as, antibodies directed against the protein may show utility as a tissue-specific marker and/or immunotherapy target for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:70 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1960 of SEQ ID NO:70, b is an integer of 15 to 1974, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:70, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 11

The translation product of this gene shares sequence homology with human squamous cell E48 antigen which is thought to be important in self-recognition and immune function.

In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: MMATPSTRPPPPAASTTSATAPALPPRPPWPWPPSSWPPSGVSSKAPEADPLKNKAL (SEQ ID NO:80); LLLTSPLPRCPPACSHDAPAHPDPGGPHGLTSGPGLGLPRVCLQRRQLLQPHALPGYGCLLH DHAHLLHPHQDEGQ (SEQ ID NO:81); and/or WLLQARVHHLLLPVRPLQRHRPCHPGHPGPGPHPPGHPLGSPLKPPRQTHSRTKLS (SEQ ID NO:82).

Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention and polynucleotides encoding these polypeptides are also encompassed by the invention.

When tested against K562 leukemia cell lines, supernatants removed from cells containing this gene activated the ISRE assay. Thus, it is likely that this gene activates leukemia cells through the Jak-STAT signal transduction pathway. The interferon-sensitive response element is a promoter element found upstream of many genes which are involved in the Jak-STAT pathway. The Jak-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jak-STAT pathway, reflected by the binding of the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells.

This gene is expressed primarily in adult brain, and to a lesser extent in fetal lung.

Polynucleotides and polypeptides of the invention would be useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, autoimmune disorders. Similarly, polypeptides and antibodies directed to these polypeptides would be useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise, or alternatively consist of one, two or all three of the immunogenic epitopes shown in SEQ ID NO:77 as residues: Tyr-28 to Phe-34, Thr-54 to Val-60, Tyr-73 to Thr-82. Polynucleotides encoding said polypeptides are encompassed by the invention.

The tissue distribution and homology to human squamous cell E48 antigen indicates that polynucleotides and polypeptides corresponding to this gene would be useful for study, diagnosis, detection, prevention and/or treatment of autoimmune diseases and disorders, such as lupus, transplant rejection, allergic reactions, and arthritis. Protein, as well as, antibodies directed against the protein may show utility as a tissue-specific marker and/or immunotherapy target for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:76 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 599 of SEQ ID NO:76, b is an integer of 15 to 613, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:76, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 12

The gene encoding the disclosed cDNA is thought to reside on chromosome 19. Accordingly, polynucleotides related to this invention would be useful as a marker in linkage analysis for chromosome 19.

The translation product of this gene is a transmembrane protein that forms disulfide-bonded homodimers and contains a motif in its cytoplasmic domain (located at the carboxy terminus of the protein relative to the transmembrane domain) that functions as an adaptor for associating protein complexes involved in triggering cellular activation. The transmembrane domain is predicted to consist of the amino acid sequence: VLAGIVMGDLVLTVLIALAVYFLG (SEQ ID NO:85).

In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: QAQSDCSCSTVSPG (SEQ ID NO:86), VLAGIVMGDLVLTVLIALAVYFLG (SEQ ID NO:87), VPRGRGAAEATRKQRITETESPYQELQGQRSDVYSDL (SEQ ID NO:88), and/or ETESPYQELQGQRSDVYSDLNT (SEQ ID NO:89). Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention and polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in macrophage, and to a lesser extent in primary dendritic cells and neutrophils.

Polynucleotides and polypeptides of the invention would be useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immunologically mediated disorders. Similarly, polypeptides and antibodies directed to these polypeptides would be useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types and cell types (e.g., blood cells, and cells and tissue of the immune system, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise, or alternatively consist of one or both of the immunogenic epitopes shown in SEQ ID NO:84 as residues: Ala-28 to Ser-33, Ala-76 to Lys-111. Polynucleotides encoding said polypeptides are encompassed by the invention.

The tissue distribution in immune tissues indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the diagnosis, treatment, and/or prevention of immune disorders including: leukemias, lymphomas, auto-immunities, immunodeficiencies (e.g., AIDS), immuno-supressive conditions (transplantation) and hematopoietic disorders. Furthermore, expression of this gene product in macrophage and primary dendritic cells also strongly indicates a role for this protein in immune function and immune surveillance. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:83 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 683 of SEQ ID NO:83, b is an integer of 15 to 697, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:83, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 13

The protein product of this gene was found to have homology to the human CD84 protein which, as a novel member of the Ig superfamily, is thought to play an important role in the modulation of the immune response. The present invention appears to encode a novel full-length CD84 homolog and is highly enriched, if not specific, for activated T cells.

In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: ITPLGLGAAD (SEQ ID NO:96), TLRVLGKVPAVCPWCALWRKAGMDMTYSWLSRGDSTYTFHEG PVLSTSWRPGDSALSYTCRANNPISNVSSCPIPDGPFYADPNYASEKPSTAFCLLAKGLLIFL LLVILAM GLWVIRVQKRHKMPRMKKLMRNRMKLRKEAKPGSSPA (SEQ ID NO:97), AVCPWCALWRKAGMDMTYSWL (SEQ ID NO:98), PGDSALSYTCRANNPISNVSSCPI (SEQ ID NO:99), YASEKPSTAFCLLAKGLLIFLLLV (SEQ ID NO:100), and/or QKRHKMPRMKKLMRNRMKLRKEAKPG (SEQ ID NO:101). Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides) are encompassed by the invention. Antibodies that bind polypeptides of the invention and polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in human activated T-Cells.

Polynucleotides and polypeptides of the invention would be useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immunodeficiencies, inflammatory conditions, infections, and other immune or hematopoietic disorders. Similarly, polypeptides and antibodies directed to these polypeptides would be useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the disorders of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise, or alternatively consist of one or both of the immunogenic epitopes shown in SEQ ID NO:91 as residues: Glu-15 to Arg-23, Asn-79 to Gly-84. Polynucleotides encoding said polypeptides are encompassed by the invention.

The tissue distribution in activated T-cells indicates that polynucleotides and polypeptides corresponding to this gene would be useful for the detection, diagnosis, treatment, and/or prevention of a variety of immune system disorders. Expression of this gene product in T-cells indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also indicate a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, and leukemia. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tumors and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:90 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1126 of SEQ ID NO:90, b is an integer of 15 to 1140, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:90, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 14

Preferred polypeptides of the present invention comprise, or alternatively consist of, one or more of the immunogenic epitopes shown in SEQ ID NO:103 as residues: Val-54 to Asp-59, Thr-55 to Leu-60 and Trp-98 to Cys-104. Polynucleotides encoding said polypeptides are also encompassed by the invention. In a specific embodiment, antibodies that bind said epitopes or other polypeptides of the invention are also encompassed by the invention.

In a specific embodiment, polypeptides of the invention, comprise or alternatively consist of, the following amino acid sequences: LSPPRGACR (SEQ ID NO:106). Polynucleotides encoding these polypeptides are also encompassed by the invention as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also preferred are polypeptides, comprising or alternatively consisting of, the mature polypeptide which is predicted to consist of residues 23-108 of the foregoing sequence (SEQ ID NO:103), and biologically active fragments of the mature polypeptide. Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

FIG. 5 show the nucleotide (SEQ ID NO:102) and deduced amino acid sequence (SEQ ID NO:103) corresponding to this gene.

FIG. 6 shows an analysis of the amino acid sequence (SEQ ID NO:103). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.

The data presented in FIG. 6 are also represented in tabular form in Table 11. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 6, and Table 11: “Res”: amino acid residue of SEQ ID NO:103 and FIG. 5; “Position”: position of the corresponding residue within SEQ ID NO:103 and FIG. 5; I: Alpha, Regions Garnier Robson; II: Alpha, Regions Chou Fasman; III: Beta, Regions Garnier Robson; IV: Beta, Regions Chou Fasman; V: Turn, Regions Garnier Robson; VI: Turn, Regions Chou Fasman; VII: Coil, Regions Garnier Robson; VIII: Hydrophilicity Plot Kyte Doolittle; IX: Hydrophobicity Plot Hopp Woods; X: Alpha, Amphipathic Regions Eisenberg; XI: Beta, Amphipathic Regions Eisenberg; XII: Flexible Regions Karplus Schulz; XIII: Antigenic Index Jameson Wolf; and XIV: Surface Probability Plot Emini.

Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consisting of, one or more of the following regions: alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in FIG. 6 and/or Table 11, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 11 can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIG. 6, but may, as shown in Table 11, be represented or identified by using tabular representations of the data presented in FIG. 6. The DNA*STAR computer algorithm used to generate FIG. 6 (set on the original default parameters) was used to present the data in FIG. 6 in a tabular format (See Table 11). The tabular format of the data in FIG. 6 is used to easily determine specific boundaries of a preferred region.

The present invention is further directed to fragments of the polynucleotide sequences described herein. By a fragment of, for example, the polynucleotide sequence of a deposited cDNA or the nucleotide sequence shown in SEQ ID NO:102, is intended polynucleotide fragments at least about 15 nt, and more preferably at least about 20 nt, at least about 25 nt, still more preferably at least about 30 nt, at least about 35 nt, and even more preferably, at least about 40 nt in length, at least about 45 nt in length, at least about 50 nt in length, at least about 60 nt in length, at least about 70 nt in length, at least about 80 nt in length, at least about 90 nt in length, at least about 100 nt in length, at least about 125 nt in length, at least about 150 nt in length, at least about 175 nt in length, which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 200-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of a deposited cDNA or as shown in SEQ ID NO:102. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:102. In this context “about” includes the particularly recited size, an sizes larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 553 of SEQ ID NO:102, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.

Preferred polypeptide fragments of the invention comprise, or alternatively consist of, the secreted protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. Particularly, N-terminal deletions of the polypeptide can be described by the general formula m-108 where m is an integer from 2 to 102, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:103. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: K-2 to P-108; A-3 to P-108; L-4 to P-108; C-5 to P-108; L-6 to P-108; L-7 to P-108; L-8 to P-108; L-9 to P-108; P-10 to P-108; V-11 to P-108; L-12 to P-108; G-13 to P-108; L-14 to P-108; L-15 to P-108; V-16 to P-108; S-17 to P-108; S-18 to P-108; K-19 to P-108; T-20 to P-108; L-21 to P-108; C-22 to P-108; S-23 to P-108; M-24 to P-108; E-25 to P-108; E-26 to P-108; A-27 to P-108; 1-28 to P-108; N-29 to P-108; E-30 to P-108; R-31 to P-108; 1-32 to P-108; Q-33 to P-108; E-34 to P-108; V-35 to P-108; A-36 to P-108; G-37 to P-108; S-38 to P-108; L-39 to P-108; 1-40 to P-108; F-41 to P-108; R-42 to P-108; A-43 to P-108; 1-44 to P-108; S-45 to P-108; S-46 to P-108; 1-47 to P-108; G-48 to P-108; L-49 to P-108; E-50 to P-108; C-51 to P-108; Q-52 to P-108; S-53 to P-108; V-54 to P-108; T-55 to P-108; S-56 to P-108; R-57 to P-108; G-58 to P-108; D-59 to P-108; L-60 to P-108; A-61 to P-108; T-62 to P-108; C-63 to P-108; P-64 to P-108; R-65 to P-108; G-66 to P-108; F-67 to P-108; A-68 to P-108; V-69 to P-108; T-70 to P-108; G-71 to P-108; C-72 to P-108; T-73 to P-108; C-74 to P-108; G-75 to P-108; S-76 to P-108; A-77 to P-108; C-78 to P-108; G-79 to P-108; S-80 to P-108; W-81 to P-108; D-82 to P-108; V-83 to P-108; R-84 to P-108; A-85 to P-108; E-86 to P-108; T-87 to P-108; T-88 to P-108; C-89 to P-108; H-90 to P-108; C-91 to P-108; Q-92 to P-108; C-93 to P-108; A-94 to P-108; G-95 to P-108; M-96 to P-108; D-97 to P-108; W-98 to P-108; T-99 to P-108; G-100 to P-108; A-101 to P-108; R-102 to P-108; and C-103 to P-108 of SEQ ID NO:103. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind ligand, ability to generate antibodies, ability to bind antibodies) may still be retained. For example the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C terminus. Whether a particular polypeptide lacking C terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a polypeptide with a large number of deleted C terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response. Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in FIG. 5 (SEQ ID NO:103), as described by the general formula 1-n, where n is an integer from 6 to 107, where n corresponds to the position of the amino acid residue identified in SEQ ID NO:103. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to Q-107; M-1 to V-106; M-1 to R-105; M-1 to C-104; M-1 to C-103; M-1 to R-102; M-1 to A-101; M-1 to G-100; M-1 to T-99; M-1 to W-98; M-1 to D-97; M-1 to M-96; M-1 to G-95; M-1 to A-94; M-1 to C-93; M-1 to Q-92; M-1 to C-91; M-1 to H-90; M-1 to C-89; M-1 to T-88; M-1 to T-87; M-1 to E-86; M-1 to A-85; M-1 to R-84; M-1 to V-83; M-1 to D-82; M-1 to W-81; M-1 to S-80; M-1 to G-79; M-1 to C-78; M-1 to A-77; M-1 to S-76; M-1 to G-75; M-1 to C-74; M-1 to T-73; M-1 to C-72; M-1 to G-71; M-1 to T-70; M-1 to V-69; M-1 to A-68; M-1 to F-67; M-1 to G-66; M-1 to R-65; M-1 to P-64; M-1 to C-63; M-1 to T-62; M-1 to A-61; M-1 to L-60; M-1 to D-59; M-1 to G-58; M-1 to R-57; M-1 to S-56; M-1 to T-55; M-1 to V-54; M-1 to S-53; M-1 to Q-52; M-1 to C-51; M-1 to E-50; M-1 to L-49; M-1 to G-48; M-1 to I-47; M-1 to S-46; M-1 to S-45; M-1 to I-44; M-1 to A-43; M-1 to R-42; M-1 to F-41; M-1 to I-40; M-1 to L-39; M-1 to S-38; M-1 to G-37; M-1 to A-36; M-1 to V-35; M-1 to E-34; M-1 to Q-33; M-1 to I-32; M-1 to R-31; M-1 to E-30; M-1 to N-29; M-1 to I-28; M-1 to A-27; M-1 to E-26; M-1 to E-25; M-1 to M-24; M-1 to S-23; M-1 to C-22; M-1 to L-21; M-1 to T-20; M-1 to K-19; M-1 to 5-18; M-1 to 5-17; M-1 to V-16; M-1 to L-15; M-1 to L-14; M-1 to G-13; M-1 to L-12; M-1 to V-11; M-1 to P-10; M-1 to L-9; M-1 to L-8; M-1 to L-7; and M-1 to L-6; of SEQ ID NO:103. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In addition, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides comprising, or alternatively consisting of, one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of SEQ ID NO:103, where n and m are integers as described above.

Also included are polynucleotide sequences encoding a polypeptide consisting of a portion of the complete amino acid sequence encoded by a cDNA clone contained in ATCC™ Deposit No. 209215, where this portion excludes any integer of amino acid residues from 1 to about 102 amino acids from the amino terminus of the complete amino acid sequence encoded by a cDNA clone contained in ATCC™ Deposit No. 209215, or any integer of amino acid residues from 6 to about 108 amino acids from the carboxy terminus, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC™ Deposit No. 209215. Polypeptides encoded by these polynucleotides also are encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Additional preferred polypeptide fragments of the invention comprise, or alternatively consist of, an amino acid sequence selected from the group: M-1 to L-15; K-2 to V-16; A-3 to S-17; L-4 to S-18; C-5 to K-19; L-6 to T-20; L-7 to L-21; L-8 to C-22; L-9 to S-23; P-10 to M-24; V-11 to E-25; L-12 to E-26; G-13 to A-27; L-14 to I-28; L-15 to N-29; V-16 to E-30; S-17 to R-31; S-18 to I-32; K-19 to Q-33; T-20 to E-34; L-21 to V-35; C-22 to A-36; S-23 to G-37; M-24 to S-38; E-25 to L-39; E-26 to I-40; A-27 to F-41; 1-28 to R-42; N-29 to A-43; E-30 to I-44; R-31 to S-45; 1-32 to S-46; Q-33 to I-47; E-34 to G-48; V-35 to L-49; A-36 to E-50; G-37 to C-51; S-38 to Q-52; L-39 to S-53; 1-40 to V-54; F-41 to T-55; R-42 to S-56; A-43 to R-57; 1-44 to G-58; S-45 to D-59; S-46 to L-60; 1-47 to A-61; G-48 to T-62; L-49 to C-63; E-50 to P-64; C-51 to R-65; Q-52 to G-66; S-53 to F-67; V-54 to A-68; T-55 to V-69; S-56 to T-70; R-57 to G-71; G-58 to C-72; D-59 to T-73; L-60 to C-74; A-61 to G-75; T-62 to S-76; C-63 to A-77; P-64 to C-78; R-65 to G-79; G-66 to 5-80; F-67 to W-81; A-68 to D-82; V-69 to V-83; T-70 to R-84; G-71 to A-85; C-72 to E-86; T-73 to T-87; C-74 to T-88; G-75 to C-89; S-76 to H-90; A-77 to C-91; C-78 to Q-92; G-79 to C-93; S-80 to A-94; W-81 to G-95; D-82 to M-96; V-83 to D-97; R-84 to W-98; A-85 to T-99; E-86 to G-100; T-87 to A-101; T-88 to R-102; C-89 to C-103; H-90 to C-104; C-91 to R-105; Q-92 to V-106; C-93 to Q-107; and A-94 to P-108 of SEQ ID NO:103. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

As described herein or otherwise known in the art, the polynucleotides of the invention have uses that include, but are not limited to, serving as probes or primers in chromosome identification, chromosome mapping, and linkage analysis.

This gene is expressed primarily in placenta and in some immune tissues and cells of the immune system (e.g., Jurkat T cell lines, and normal bone marrow).

Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, fetal deficiencies, pre-natal disorders, and vascular diseases and conditions. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., developmental, proliferating, vascular, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution in placenta indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of developmental anomalies, fetal deficiencies, reproductive disfunction or pre-natal disorders. Moreover, the protein is useful in the detection, treatment, and/or prevention of a variety of vascular disorders and conditions, which include, but are not limited to miscrovascular disease, vascular leak syndrome, aneurysm, stroke, embolism, thrombosis, coronary artery disease, arteriosclerosis, and/or atherosclerosis. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

The tissue distribution of the expression of this gene indicates that polynucleotides and polypeptides corresponding to this gene (as well as antibodies raised against those polypeptides) are useful for the diagnosis and treatment of diseases and disorders associated with the immune system, including, but not limited to, allergy, asthma, graft rejection, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and other auotimmune conditions, infections, AIDS, chronic variable immune deficiency (CVID) and other immune deficiency syndromes, respiratory distress syndrome and inflammation, neoplasms of the immune/hematopoetic system including leukemias, lymphomas and other proliferative disorders such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma, and myelodypsplastic syndromes. The polynucleotides and/or polypeptides corresponding to this gene (and/or antibodies raised against those polypeptides) may also be useful for stimulating the immune response to bolster the immune response to diseases such as cancer or infection.

Furthermore, the protein may also be used to determine unknown biological activities, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, are used to treat, prevent, and/or diagnose diseases and disorders of the endocrine system.

In specific embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to diagnose, prognose or monitor vascular diseases. In other specifc embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to treat, prevent, or ameliorate vascular diseases

In other preferred embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to diagnose, prognose or monitor diseases and disorders associated with aberrant glucose metabolism or glucose uptake into cells. In other specifc embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to treat, prevent, or ameliorate diseases and disorders associated with aberrant glucose metabolism or glucose uptake into cells.

It is believed that increased expression of this gene, at either the RNA or protein level, is increased in individuals (or a subset of individuals) that either have a predisposition to develop or have already developed type II diabetes mellitus (non-insulin dependent diabetes). Thus, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to diagnose, prognose, and/or monitor individuals with type II diabetes mellitus or individuals with a predisposition to develop type II diabetes mellitus.

By “agonist,” is meant any substance that enhances the function of the polynucleotides or polypeptides of the invention. Classes of molecules that can function as agonists include, but are not limited to, small molecules, antibodies (including fragments or variants thereof, such as Fab fragments, Fab′2 fragments and scFvs), and peptidomimetics. By “antagonist,” is meant any substance that diminishes or abolishes the function of the polynucleotides or polypeptides of the invention. Classes of molecules that can function as antagonists include, but are not limited to, small molecules, antibodies (including fragments or variants thereof, such as Fab fragments, Fab′2 fragments and scFvs) antisense polynucleotides, ribozymes, and peptidomimetics.

A biological sample of persons afflicted with type II diabetes mellitus is believed to be characterized by high levels of expression of this gene when compared to that observed in individuals not having type II diabetes mellitus. Thus, polynucleotides and/or polypeptides of the invention, and/or agonists or antagonists thereof, may be used according to the methods of the invention in the diagnosis and/or prognosis of individuals with type II diabetes mellitus, a subset of individuals with type II diabetes mellitus, and/or individuals or a subset of individuals with a predisposition to develop type II diabetes mellitus. For example, a biological sample obtained from a person suspected of being afflicted with type II diabetes mellitus, “the subject,” may be analyzed for the relative expression level(s) of polynucleotides and/or polypeptides of the invention. The expression level(s) of one or more of these molecules of the invention is (are) then compared to the expression level(s) of the same molecules of the invention as expressed in a person known not to be afflicted with rheumatoid arthritis. An increase in the expression level(s) of this gene in samples obtained from the subject compared to the control suggests that the subject is afflicted with type II diabetes mellitus or a subset thereof.

In another embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or antagonists thereof (especially neutralizing or antagonistic antibodies) may be used to treat, prevent, and/or ameliorate type II diabetes. Additionally, in other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or anatgonists thereof (especially neutralizing or antagonistic antibodies) may be used to treat, prevent, or ameliorate conditions associated with type II diabetes mellitus, including, but not limited to, seizures, mental confusion, drowsiness, nonketotic hyperglycemic-hyperosmolar coma, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the “Cardiovascular Disorders” section below), dyslipidemia, kidney disease (e.g., renal failure, nephropathy other diseases and disorders as described in the “Renal Disorders” section below), nerve damage, neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, infections (e.g., infectious diseases and disorders as described in the “Infectious Diseases” section below, especially of the urinary tract and skin), carpal tunnel syndrome and Dupuytren's contracture.

In other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to regulate the animal's weight. In specific embodiments the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to control the animal's weight by modulating a biochemical pathway involving insulin. In still other embodiments the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to control the animal's weight by modulating a biochemical pathway involving insulin-like growth factor.

In a preferred embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to treat weight disorders, including but not limited to, obesity, cachexia, wasting disease, anorexia, and bulimia.

In other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are useful for the treatment, prevention or amelioration of neurodegenerative disorders including, but not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations, and other neurolgical diseases and disorders as described in the “Neural Activity and Neurological Activity diseases” section below.

In another embodiment, compositions of the invention (comprising polynucleotides, polypeptides of the invention, agonists and/or antagonists thereof (including antibodies) as well as fragments and variants of the polynucleotides, polypeptides of the invention, agonists and/or antagonists of the invention) are used in combination with anti-diabetic drugs. In a specific embodiment, compositions of the invention are administered in combination with thiazolidinediones (TZDs) including, but not limited to, rosiglitazone, piogliatazone, and troglitazone. In another specific embodiment, compositions of the invention are used in combination with oral hypoglycemic sulfonylurea drugs including, but not limited to, acarbose, acetohexamide, chlorpropamide, glimepiride, glipizide, glyburide, metformin, tolazamide, and/or tolbutamide. In still other embodiments, compositions of the invention are administered in combination with one or more of the following: a biguanide antidiabetic agent, a glitazone antidiabetic agent, and a sulfonylurea antidiabetic agent.

Features of Protein Encoded by Gene No: 15

The translation product of this gene shares sequence homology with drosophila peroxidasin which is thought to be important in extracellular matrix architecture. Moreover, the protein has homology to receptor-linked protein tyrosine phosphatases, which play important roles in inflammatory diseases and immune disorders. When tested against Jurkat T-cell lines, supernatants removed from cells containing this gene activated the GAS pathway. Thus, it is likely that this gene activates T-cells through the Jaks-STAT signal transduction pathway. GAS is a promoter element found upstream in many genes which are involved in the Jaks-STAT pathway. The Jaks-STAT pathway is a large, signal transduction pathway involved in the differentiation and proliferation of cells. Therefore, activation of the Jaks-STAT pathway, reflected by the binding of the GAS element, can be used to indicate proteins involved in the proliferation and differentiation of cells.

In specific embodiments, polypeptides of the invention comprise the following amino acid sequence: GRPTRPLRVA (SEQ ID NO:111), AWCPQTHTTSCLMGPFCCYSPLPGDMPTMARPCPQTWVSTHVRPATGLARQSAEALGCL WLSSGRISRSSLGTWWLWWVSSLLWNVGRPGATQSPQSHGGKMGNPWPSSPEGTQCPGG PC (SEQ ID NO:112), CCYSPLPGDMPTMARPCPQTWVSTH (SEQ ID NO:113), ALGC LWLSSGRISRSSLG (SEQ ID NO:114), and/or WNVGRPGATQSPQSHG GKMGNPWPSSPE (SEQ ID NO:115). Polynucleotides encoding these polypeptides are also encompassed by the invention.

This gene is expressed primarily in umbilical vein and to a lesser extent in endothelial and brain cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune, developmental and growth diseases and/or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of fetal tissues, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, neural, cancerous and wounded tissues) or bodily fluids (e.g. lymph, amniotic fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Preferred epitopes include those comprising a sequence shown in SEQ ID NO:108 as residues: Ala-55 to Thr-62, His-164 to Gly-175, Ala-197 to Glu-202.

The tissue distribution in umbilical vein cells, and homology to peroxidasin and receptor-linked protein tyrosine phosphatases indicates that polynucleotides and polypeptides corresponding to this gene are useful for the study, diagnosis, and treatment of various fetal developmental and growth disorders involving the formation of extracellular matrix. Alternatively, the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of a variety of immune system disorders. Activation of the GAS pathway by the gene product indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells.

This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses). Since the gene product demonstrates activity with regard to the GAS pathway, the natural gene product may be involved in immune functions. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis, and tissues. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Protein is useful in modulating the immune response to proliferative and vascular cells and tissues, particularly those having aberrant phenotypes. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Features of Protein Encoded by Gene No: 16

The translation product of this gene shares sequence homology with the kidney injury molecule-1 of Rattus norvegicus which is thought to play an important role in the restoration of the morphological integrity and function to postischemic kidney (See Genbank Accession No. gi|2665892 (AF035963)). Polynucleotides and polypeptides of the present invention are useful to promote growth of new tissue and survival of damaged tissue. Recombinant polypeptides of the present invention can be expressed in prokaryotic and eukaryotic host cells using a claimed process. Soluble variants fused to a toxin, imageable compound or radionuclide, and IgG fusion proteins are also claimed.

Polynucleotides and polypeptides of the present invention or an agonist, can be used to treat renal disease and to promote the growth of new tissue or the survival of damaged tissue, generally in conditions where the binding of specific ligand to the present invention stimulates cell growth, maintains cellular differentiation or reduces apoptosis, e.g. in cases of renal failure, nephritis, kidney transplants, toxic or hypoxic injury. A monoclonal antibody specific for polynucleotides and polypeptides of the present invention can be used to treat renal disease, e.g. where binding of the invention to a ligand results in neoplasia, loss of cellular function, susceptibility to apoptosis or promotion of inflammation, deliver imaging agents to the cells expressing the present invention in vivo or in vitro and measure the concentration of the present invention by immunoassay. Damage/regeneration of renal cells can be determined by measuring the present invention, particularly to diagnose or monitor the progress of disease or therapy. The tumour cells expressing the present invention can be inhibited by treatment with a fusion protein comprising a ligand of the present invention or MAb with a toxin or radionuclide, and tumour cells that express the present invention ligand can be inhibited with similarly tagged polypeptides of the present invention or anti-present invention ligand antibody.

Preferred polypeptides of the invention comprise the following amino acid sequence: HESTVK (SEQ ID NO:122). Polynucleotides encoding these polypeptides are also provided.

This gene is expressed primarily in infant brain and fetal liver, and to a lesser extent in neoplastic cell lines and endocrine organs.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions, which include, but are not limited to, immune, neural, endorcine, and growth disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune and endocrine systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, neural, endorcine, growth, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, amniotic fluid, bile, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO:117 as residues: Ser-44 to Ser-51, Cys-53 to Cys-64, Val-76 to Lys-83, Pro-102 to Gly-108, Arg-133 to Thr-162, Thr-169 to Lys-183. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in fetal liver and infant brain indicates that polynucleotides and polypeptides corresponding to this gene are useful for study and treatment of immune and developmental conditions. Moreover, the expression within infant and fetal tissues and other cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis and treatment of cancer and other proliferative disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51, and elsewhere herein. Similarly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation. Thus this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Alternatively, the protein product of this gene could be used in the treatment and/or detection of kidney diseases including renal failure, nephritus, renal tubular acidosis, proteinuria, pyuria, edema, pyelonephritis, hydronephritis, nephrotic syndrome, crush syndrome, glomerulonephritis, hematuria, renal colic and kidney stones, in addition to Wilm's Tumor Disease, and congenital kidney abnormalities such as horseshoe kidney, polycystic kidney, and Falconi's syndrome. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:116 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 557 of SEQ ID NO:116, b is an integer of 15 to 571, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:116, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 17

The translation product of this gene shares some sequence homology with various chains of the T-cell receptor, which are important in signalling between different cells of the immune system.

The gene encoding the disclosed cDNA is thought to reside on the X chromosome. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for the X chromosome.

This gene is expressed primarily in placental tissue, and to a lesser extent in activated monocytes and dendritic cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune disorders and reproductive disorders, particularly pregnancy-associated disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system and female reproductive system, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, reproductive, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO:124 as residues: Val-29 to Val-37, Asp-71 to His-76, Gln-78 to Gly-84, Met-105 to His-110, Trp-117 to Gly-122, Gln-136 to Lys-141, Leu-143 to Ala-149, Thr-162 to Asp-174, Ser-181 to Lys-186, Arg-214 to Glu-220, Glu-232 to Glu-238, Cys-249 to Asp-265. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in dendritic cells, activated monocytes and placental tissue (a tissue rich in hematopoeitic cells), and its homology to the T-cell receptor, indicates that polynucleotides and polypeptides corresponding to this gene are useful in the treatment, prophylaxis and/or diagnosis of immune and autoimmune diseases, such as lupus, transplant rejection, allergic reactions, arthritis, asthma, immunodeficiency diseases, leukemia, and AIDS. Its expression predominantly in hematopoietic cells also indicates that the gene could be important for the treatment and/or detection of hematopoietic disorders such as graft versus host reaction, graft versus host disease, transplant rejection, myelogenous leukemia, bone marrow fibrosis, and myeloproliferative disease. The protein could also be used to enhance or protect the proliferation, differentiation, and functional activation of hematopoietic progenitor cells such as bone marrow cells, which could be useful for cancer patients undergoing chemotherapy or patients undergoing bone marrow transplantation.

The protein may also be useful as a means to increase the proliferation of peripheral blood leukocytes, which could be useful in the combat of a range of hematopoietic disorders including immunodeficiency diseases, leukemia, and septicemia. In addition, expression in placenta indicates the gene or the protein encoded by this gene could be useful in the treatment, prophylaxis and/or diagnosis of placentitis, placenta previa, pregnancy disease, and miscarriage. Specific expression within the placenta indicates that this gene product may play a role in the proper establishment and maintenance of placental function. Alternately, this gene product is produced by the placenta and then transported to the embryo, where it may play a crucial role in the development and/or survival of the developing embryo or fetus. Expression of this gene product in a vascular-rich tissue such as the placenta also indicates that this gene product is produced more generally in endothelial cells or within the circulation. In such instances, it may play more generalized roles in vascular function, such as in angiogenesis. It may also be produced in the vasculature and have effects on other cells within the circulation, such as hematopoietic cells. It may serve to promote the proliferation, survival, activation, and/or differentiation of hematopoietic cells, as well as other cells throughout the body. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:123 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1576 of SEQ ID NO:123, b is an integer of 15 to 1590, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:123, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 18

This gene is expressed primarily in adipose tissue and dendritic cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, metabolic and immune disorders or diseases, particularly obesity. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune, metabolic and digestive systems, expression of this gene at significantly higher or lower levels is routinely detected in certain tissues or cell types (e.g., immune, metabolic, digestive, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO:128 as residues: Ile-40 to Glu-45, Cys-63 to Val-69, Glu-83 to Asn-94, Pro-107 to Cys-115, Phe-137 to Ser-143, Ser-159 to Thr-167, Glu-200 to Tyr-210. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in primarily adipose tissue indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment, diagnosis and/or prophylaxis of obesity related disorders. In addition, expression in dendritic cells indicates a potential role in the treatment, diagnosis and/or prophylaxis of immune and autoimmune disorders such as lupus, transplant rejection, allergic reactions, arthritis, asthma, immunodeficiency diseases, leukemia, and AIDS. The tissue distribution in adipose tissue indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment of obesity and other metabolic and endocrine conditions or disorders. Furthermore, the protein product of this gene may show utility in ameliorating conditions which occur secondary to aberrant fatty-acid metabolism (e.g. aberrant myelin sheath development), either directly or indirectly. Expression of this gene product in dendritic cells indicates a role in the regulation of the proliferation; survival; differentiation; and/or activation of potentially all hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g. by boosting immune responses).

Since the gene is expressed in cells of lymphoid origin, the gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it is also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Expression of this gene product in dendritic cells also strongly indicates a role for this protein in immune function and immune surveillance. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:127 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1889 of SEQ ID NO:127, b is an integer of 15 to 1903, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:127, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 19

The translation product of this gene shares sequence homology with alpha-1 antitrypsin (See Genebank accession no. gn1|PID|d1021080 and BAA20264; all references available through this accession are hereby incorporated by reference herein). Alpha-1-antitrypsin is an important plasma protease inhibitor affecting a wide variety of serine proteases involved in coagulation, fibrinolysis and kinen generation.

In specific embodiments, polypeptides of the invention comprise, or alternatively consists of, the following amino acid sequence: GERRNWGGEVYYSTGYSSRK (SEQ ID NO:133). Moreover, fragments and variants of this polypeptide (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding this polypeptide are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding said polypeptides are also encompassed by the invention.

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 22-414 of the amino acid sequence referenced in Table 1A for this gene. Moreover, a cytoplasmic tail encompassing amino acids 5-21 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ib membrane proteins.

This gene is expressed in healing groin wound and to a lesser extent in some other tissues.

Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, wound healing disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the healing groin wound, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., healing, regenerative, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Preferred polypeptides of the present invention comprise, or alternatively consist of, one or more immunogenic epitopes shown in SEQ ID NO:132 as residues: Phe-25 to Tyr-30, Gln-37 to Arg-42, Lys-106 to Leu-112, Leu-123 to Leu-130, Gln-142 to Phe-150, Gln-183 to Lys-188, Asp-219 to Glu-226, Lys-359 to Glu-366. Polynucleotides encoding said polypeptides are also encompassed by the invention.

The tissue distribution in healing groin wound and homology to alpha-1 antitrypsin indicates that polynucleotides and polypeptides corresponding to this gene are useful for diagnosis and therapeutic treatment of wound healing disorders. In addition, since healing wounds have transcriptional environments similar to developing tissues, the translation product of this gene may be useful for the diagnosis and treatment of cancer and other proliferative disorders.

Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Features of Protein Encoded by Gene No: 20

This gene is expressed in cerebellum and ovarian cancer.

Preferred polypeptides of the present invention comprise, or alternatively consist of, one or more of the immunogenic epitopes shown in SEQ ID NO:135 as residues: Thr-41 to Gly-47, Pro-170 to Asp-176, Leu-257 to Trp-262, Gln-276 to Ser-283, Arg-323 to Leu-330, Pro-409 to Ser-427, Gly 440 to Ala-449, Arg-323 to Gly-331, Glu-348 to Ser 354, Arg-256 to Trp-262, Phe 278 to Val 285, Arg 362 to Gly 385. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO:140 as residues: Thr-41 to Gly-47, Pro-170 to Asp-176, Leu-257 to Trp-262, Gln-276 to Ser-283, Arg-323 to Leu-330, Pro-362 to Val-374. Polynucleotides encoding said polypeptides are also encompassed by the invention. Antibodies that bind said epitopes or other polypeptides of the invention are also encompassed.

In a specific embodiment, polypeptides of the invention, comprise or alternatively consist of, an amino acid sequence selected from the group: FHGLGRLHTVHL (SEQ ID NO:141), AAFTGLALLEQLDLSDNAQLR (SEQ ID NO:142), HEVPDAPRPTPT (SEQ ID NO:143), and/or AFRGLHSLD (SEQ ID NO:144). Polynucleotides encoding these polypeptides are also encompassed by the invention as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also preferred are polypeptides, comprising or alternatively consisting of, the mature polypeptide which is predicted to consist of residues 27-473 of the foregoing sequence (SEQ ID NO:135), and biologically active fragments of the mature polypeptide (e.g., fragments that prevent neural/neuronal damage and/or spinal cord injury). Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In a nonexclusive specific embodiment, polypeptides of the invention, comprise or alternatively consist of, one or more of the following amino acid sequences and biologically active fragments thereof (e.g., fragments that prevent neural/neuronal damage and/or spinal cord injury).: SQRIFLHGNRISHVP AASFRAC (SEQ ID NO:145), LTILWLHSNVLARIDAAAFTGL (SEQ ID NO:146), LEQLDLSDNAQLRSVDPATFHGL (SEQ ID NO:147), LHTLHLDRC GLQELGPGLFRGL (SEQ ID NO:148), LQYLYLQDNALQALPDDTFRDL (SEQ ID NO:149), LTHLFLHGNRISSVPERAFRGL (SEQ ID NO:150), LDRLLLH QNRVAHVHPHAFRDL (SEQ ID NO:151), LMTLYLFANNLSALPTEALAPL (SEQ ID NO:152), AHCSAARGLRATR (SEQ ID NO:153), PAHCSAARGLRATRF (SEQ ID NO:154), PSLTCSLTPLGLALVLWTVLGPC (SEQ ID NO:155), LPSLTCSLTPLGLALVLWTVL (SEQ ID NO:156), LPSLTCSLTPLGLALVLWTVLGPC (SEQ ID NO:157), and CRNLTILWLHSNVL (SEQ ID NO:158). Polynucleotides encoding these polypeptides are also encompassed by the invention as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In a further embodiment of the invention, a Fc region of an immunoglobulin (e.g., IgG Fc) molecule is fused to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or all fourteen of the polypeptide(s) selected from the group consisting of: SQRIFLHGNRISHVPAASFRAC (SEQ ID NO:145), LTILWLHSNVLARIDAAAFTGL (SEQ ID NO:146), LEQLDLSDNAQLRS VDPATFHGL (SEQ ID NO:147), LHTLHLDRCGLQELGPGLFRGL (SEQ ID NO:148), LQYLYLQDNALQALPDDTFRDL (SEQ ID NO:149), LTHLFLH GNRISSVPERAFRGL (SEQ ID NO:150), LDRLLLHQNRVAHVHPHAFRDL (SEQ ID NO:151), LMTLYLFANNLSALPTEALAPL (SEQ ID NO:152), AHCSAARGLRATR(SEQ ID NO:153), PAHCSAARGLRATRF (SEQ ID NO:154), PSLTCSLTPLGLALVLWTVLGPC (SEQ ID NO:155), LPSLTCSLTPLGLA LVLWTVL (SEQ ID NO:156), LPSLTCSLTPLGLALVLWTVLGPC (SEQ ID NO:157), and CRNLTILWLHSNVL (SEQ ID NO:158). Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In additional embodiments, proteins comprising fragments or variants of a polypeptide of SEQ ID NO:135 (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement there of) are fused to an Fc region of an immunoglobulin that bind the 66-amino acid extracellular loop of Nogo-A (See Genbank Accession CAB99248; GrandPre, T., et al., Nature, 403, pp. 439-444 (2000). Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides.

Thus, the fragment, derivative or analog of the polypeptide of FIGS. 7A-C (SEQ ID NO:135), or that encoded by the deposited cDNA plasmid, may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the extracellular domain of the polypeptide is fused with another compound, such as a compound to increase the half life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the extracellular domain of the polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the extracellular domain of the polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (said references incorporated by reference in their entireties).

As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention (e.g., those comprising an immunogenic or antigenic epitope) can be fused to heterologous polypeptide sequences. For example, polypeptides of the present invention (including fragments or variants thereof), may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides. By way of another non-limiting example, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Pat. No. 5,766,883 herein incorporated by reference in its entirety. Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused to either the N- or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide). Polynucleotides encoding fusion proteins of the invention are also encompassed by the invention.

Such fusion proteins as those described above may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ° HAD tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

FIGS. 7A-C show the nucleotide (SEQ ID NO:134) and deduced amino acid sequence (SEQ ID NO:135) corresponding to this gene.

FIG. 8 shows an analysis of the amino acid sequence (SEQ ID NO:135). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings of the recited computer algorithms. In the “Antigenic Index or Jameson Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. Polypeptides comprising, or alternatively consisting of, domains defined by these graphs are contemplated by the present invention, as are polynucleotides encoding these polypeptides.

The data presented in FIG. 8 are also represented in tabular form in Table 9. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 8, and Table 9: “Res”: amino acid residue of SEQ ID NO:135 and FIGS. 7A-C; “Position”: position of the corresponding residue within SEQ ID NO:135 and FIGS. 7A-C; I: Alpha, Regions Garnier Robson; II: Alpha, Regions Chou Fasman; III: Beta, Regions Garnier Robson; IV: Beta, Regions Chou Fasman; V: Turn, Regions Garnier Robson; VI: Turn, Regions Chou Fasman; VII: Coil, Regions Garnier Robson; VIII: Hydrophilicity Plot Kyte Doolittle; IX: Hydrophobicity Plot Hopp Woods; X: Alpha, Amphipathic Regions Eisenberg; XI: Beta, Amphipathic Regions Eisenberg; XII: Flexible Regions Karplus Schulz; XIII: Antigenic Index Jameson Wolf; and XIV: Surface Probability Plot Emini.

Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consisting of, one or more of the following regions: alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in FIG. 8 and/or Table 9, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 9 can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIG. 8, but may, as shown in Table 9, be represented or identified by using tabular representations of the data presented in FIG. 8. The DNA*STAR computer algorithm used to generate FIG. 8 (set on the original default parameters) was used to present the data in FIG. 8 in a tabular format (See Table 9). The tabular format of the data in FIG. 8 is used to easily determine specific boundaries of a preferred region.

The present invention is further directed to fragments of the polynucleotide sequences described herein. By a fragment of, for example, the polynucleotide sequence of a deposited cDNA or the nucleotide sequence shown in SEQ ID NO:134, is intended polynucleotide fragments at least about 15 nt, and more preferably at least about 20 nt, at least about 25 nt, still more preferably at least about 30 nt, at least about 35 nt, and even more preferably, at least about 40 nt in length, at least about 45 nt in length, at least about 50 nt in length, at least about 60 nt in length, at least about 70 nt in length, at least about 80 nt in length, at least about 90 nt in length, at least about 100 nt in length, at least about 125 nt in length, at least about 150 nt in length, at least about 175 nt in length, which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 200-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of a deposited cDNA or as shown in SEQ ID NO:134. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:134. In this context “about” includes the particularly recited size, an sizes larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150 from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1400, from about 1401 to about 1450, and from about 1451 to about 1500, from about 1501 to about 1550, from about 1551 to about 1600, from about 1601 to about 1650 from about 1651 to about 1700, from about 1701 to about 1750, from about 1751 to about 1777 of SEQ ID NO:134, or the complementary strand thereto, or the cDNA contained in a deposited clone. In this context “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.

Preferred polypeptide fragments of the invention comprise, or alternatively consist of, the secreted protein having a continuous series of deleted residues from the amino or the carboxy terminus, or both. Particularly, N-terminal deletions of the polypeptide can be described by the general formula m-473 where m is an integer from 2 to 467, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:135. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: K-2 to C-473; R-3 to C-473; A-4 to C-473; S-5 to C-473; A-6 to C-473; G-7 to C-473; G-8 to C-473; S-9 to C-473; R-10 to C-473; L-11 to C-473; L-12 to C-473; A-13 to C-473; W-14 to C-473; V-15 to C-473; L-16 to C-473; W-17 to C-473; L-18 to C-473; Q-19 to C-473; A-20 to C-473; W-21 to C-473; Q-22 to C-473; V-23 to C-473; A-24 to C-473; A-25 to C-473; P-26 to C-473; C-27 to C-473; P-28 to C-473; G-29 to C-473; A-30 to C-473; C-31 to C-473; V-32 to C-473; C-33 to C-473; Y-34 to C-473; N-35 to C-473; E-36 to C-473; P-37 to C-473; K-38 to C-473; V-39 to C-473; T-40 to C-473; T-41 to C-473; S-42 to C-473; C-43 to C-473; P-44 to C-473; Q-45 to C-473; Q-46 to C-473; G-47 to C-473; L-48 to C-473; Q-49 to C-473; A-50 to C-473; V-51 to C-473; P-52 to C-473; V-53 to C-473; G-54 to C-473; 1-55 to C-473; P-56 to C-473; A-57 to C-473; A-58 to C-473; S-59 to C-473; Q-60 to C-473; R-61 to C-473; 1-62 to C-473; F-63 to C-473; L-64 to C-473; H-65 to C-473; G-66 to C-473; N-67 to C-473; R-68 to C-473; 1-69 to C-473; S-70 to C-473; H-71 to C-473; V-72 to C-473; P-73 to C-473; A-74 to C-473; A-75 to C-473; S-76 to C-473; F-77 to C-473; R-78 to C-473; A-79 to C-473; C-80 to C-473; R-81 to C-473; N-82 to C-473; L-83 to C-473; T-84 to C-473; 1-85 to C-473; L-86 to C-473; W-87 to C-473; L-88 to C-473; H-89 to C-473; S-90 to C-473; N-91 to C-473; V-92 to C-473; L-93 to C-473; A-94 to C-473; R-95 to C-473; 1-96 to C-473; D-97 to C-473; A-98 to C-473; A-99 to C-473; A-100 to C-473; F-101 to C-473; T-102 to C-473; G-103 to C-473; L-104 to C-473; A-105 to C-473; L-106 to C-473; L-107 to C-473; E-108 to C-473; Q-109 to C-473; L-110 to C-473; D-111 to C-473; L-112 to C-473; S-113 to C-473; D-114 to C-473; N-115 to C-473; A-116 to C-473; Q-117 to C-473; L-118 to C-473; R-119 to C-473; S-120 to C-473; V-121 to C-473; D-122 to C-473; P-123 to C-473; A-124 to C-473; T-125 to C-473; F-126 to C-473; H-127 to C-473; G-128 to C-473; L-129 to C-473; G-130 to C-473; R-131 to C-473; L-132 to C-473; H-133 to C-473; T-134 to C-473; L-135 to C-473; H-136 to C-473; L-137 to C-473; D-138 to C-473; R-139 to C-473; C-140 to C-473; G-141 to C-473; L-142 to C-473; Q-143 to C-473; E-144 to C-473; L-145 to C-473; G-146 to C-473; P-147 to C-473; G-148 to C-473; L-149 to C-473; F-150 to C-473; R-151 to C-473; G-152 to C-473; L-153 to C-473; A-154 to C-473; A-155 to C-473; L-156 to C-473; Q-157 to C-473; Y-158 to C-473; L-159 to C-473; Y-160 to C-473; L-161 to C-473; Q-162 to C-473; D-163 to C-473; N-164 to C-473; A-165 to C-473; L-166 to C-473; Q-167 to C-473; A-168 to C-473; L-169 to C-473; P-170 to C-473; D-171 to C-473; D-172 to C-473; T-173 to C-473; F-174 to C-473; R-175 to C-473; D-176 to C-473; L-177 to C-473; G-178 to C-473; N-179 to C-473; L-180 to C-473; T-181 to C-473; H-182 to C-473; L-183 to C-473; F-184 to C-473; L-185 to C-473; H-186 to C-473; G-187 to C-473; N-188 to C-473; R-189 to C-473; 1-190 to C-473; S-191 to C-473; S-192 to C-473; V-193 to C-473; P-194 to C-473; E-195 to C-473; R-196 to C-473; A-197 to C-473; F-198 to C-473; R-199 to C-473; G-200 to C-473; L-201 to C-473; H-202 to C-473; S-203 to C-473; L-204 to C-473; D-205 to C-473; R-206 to C-473; L-207 to C-473; L-208 to C-473; L-209 to C-473; H-210 to C-473; Q-211 to C-473; N-212 to C-473; R-213 to C-473; V-214 to C-473; A-215 to C-473; H-216 to C-473; V-217 to C-473; H-218 to C-473; P-219 to C-473; H-220 to C-473; A-221 to C-473; F-222 to C-473; R-223 to C-473; D-224 to C-473; L-225 to C-473; G-226 to C-473; R-227 to C-473; L-228 to C-473; M-229 to C-473; T-230 to C-473; L-231 to C-473; Y-232 to C-473; L-233 to C-473; F-234 to C-473; A-235 to C-473; N-236 to C-473; N-237 to C-473; L-238 to C-473; S-239 to C-473; A-240 to C-473; L-241 to C-473; P-242 to C-473; T-243 to C-473; E-244 to C-473; A-245 to C-473; L-246 to C-473; A-247 to C-473; P-248 to C-473; L-249 to C-473; R-250 to C-473; A-251 to C-473; L-252 to C-473; Q-253 to C-473; Y-254 to C-473; L-255 to C-473; R-256 to C-473; L-257 to C-473; N-258 to C-473; D-259 to C-473; N-260 to C-473; P-261 to C-473; W-262 to C-473; V-263 to C-473; C-264 to C-473; D-265 to C-473; C-266 to C-473; R-267 to C-473; A-268 to C-473; R-269 to C-473; P-270 to C-473; L-271 to C-473; W-272 to C-473; A-273 to C-473; W-274 to C-473; L-275 to C-473; Q-276 to C-473; K-277 to C-473; F-278 to C-473; R-279 to C-473; G-280 to C-473; S-281 to C-473; S-282 to C-473; S-283 to C-473; E-284 to C-473; V-285 to C-473; P-286 to C-473; C-287 to C-473; S-288 to C-473; L-289 to C-473; P-290 to C-473; Q-291 to C-473; R-292 to C-473; L-293 to C-473; A-294 to C-473; G-295 to C-473; R-296 to C-473; D-297 to C-473; L-298 to C-473; K-299 to C-473; R-300 to C-473; L-301 to C-473; A-302 to C-473; A-303 to C-473; N-304 to C-473; D-305 to C-473; L-306 to C-473; Q-307 to C-473; G-308 to C-473; C-309 to C-473; A-310 to C-473; V-311 to C-473; A-312 to C-473; T-313 to C-473; G-314 to C-473; P-315 to C-473; Y-316 to C-473; H-317 to C-473; P-318 to C-473; 1-319 to C-473; W-320 to C-473; T-321 to C-473; G-322 to C-473; R-323 to C-473; A-324 to C-473; T-325 to C-473; D-326 to C-473; E-327 to C-473; E-328 to C-473; P-329 to C-473; L-330 to C-473; G-331 to C-473; L-332 to C-473; P-333 to C-473; K-334 to C-473; C-335 to C-473; C-336 to C-473; Q-337 to C-473; P-338 to C-473; D-339 to C-473; A-340 to C-473; A-341 to C-473; D-342 to C-473; K-343 to C-473; A-344 to C-473; S-345 to C-473; V-346 to C-473; L-347 to C-473; E-348 to C-473; P-349 to C-473; G-350 to C-473; R-351 to C-473; P-352 to C-473; A-353 to C-473; S-354 to C-473; A-355 to C-473; G-356 to C-473; N-357 to C-473; A-358 to C-473; L-359 to C-473; K-360 to C-473; G-361 to C-473; R-362 to C-473; V-363 to C-473; P-364 to C-473; P-365 to C-473; G-366 to C-473; D-367 to C-473; S-368 to C-473; P-369 to C-473; P-370 to C-473; G-371 to C-473; N-372 to C-473; G-373 to C-473; S-374 to C-473; G-375 to C-473; P-376 to C-473; R-377 to C-473; H-378 to C-473; 1-379 to C-473; N-380 to C-473; D-381 to C-473; S-382 to C-473; P-383 to C-473; F-384 to C-473; G-385 to C-473; T-386 to C-473; L-387 to C-473; P-388 to C-473; G-389 to C-473; S-390 to C-473; A-391 to C-473; E-392 to C-473; P-393 to C-473; P-394 to C-473; A-395 to C-473; H-396 to C-473; C-397 to C-473; S-398 to C-473; A-399 to C-473; A-400 to C-473; R-401 to C-473; G-402 to C-473; L-403 to C-473; R-404 to C-473; A-405 to C-473; T-406 to C-473; R-407 to C-473; F-408 to C-473; P-409 to C-473; T-410 to C-473; S-411 to C-473; G-412 to C-473; P-413 to C-473; R-414 to C-473; R-415 to C-473; R-416 to C-473; P-417 to C-473; G-418 to C-473; C-419 to C-473; S-420 to C-473; R-421 to C-473; K-422 to C-473; N-423 to C-473; R-424 to C-473; T-425 to C-473; R-426 to C-473; S-427 to C-473; H-428 to C-473; C-429 to C-473; R-430 to C-473; L-431 to C-473; G-432 to C-473; Q-433 to C-473; A-434 to C-473; G-435 to C-473; S-436 to C-473; G-437 to C-473; G-438 to C-473; G-439 to C-473; G-440 to C-473; T-441 to C-473; G-442 to C-473; D-443 to C-473; S-444 to C-473; E-445 to C-473; G-446 to C-473; S-447 to C-473; G-448 to C-473; A-449 to C-473; L-450 to C-473; P-451 to C-473; S-452 to C-473; L-453 to C-473; T-454 to C-473; C-455 to C-473; S-456 to C-473; L-457 to C-473; T-458 to C-473; P-459 to C-473; L-460 to C-473; G-461 to C-473; L-462 to C-473; A-463 to C-473; L-464 to C-473; V-465 to C-473; L-466 to C-473; W-467 to C-473; and T-468 to C-473 of SEQ ID NO:135. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C terminus of a protein results in modification of loss of one or more biological functions of the protein (e.g., modulation of neurite growth, including, but not limited to, inhibitory activity and/or antiinflammatory activity), other functional activities (e.g., biological activities, ability to multimerize, ability to bind ligand, ability to generate antibodies, ability to bind antibodies) may still be retained. For example the ability of the shortened polypeptide to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C terminus. Whether a particular polypeptide lacking C terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a polypeptide with a large number of deleted C terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in FIGS. 7A-C (SEQ ID NO:135), as described by the general formula 1-n, where n is an integer from 6 to 472, where n corresponds to the position of the amino acid residue identified in SEQ ID NO:135. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to P-472; M-1 to G-471; M-1 to L-470; M-1 to V-469; M-1 to T-468; M-1 to W-467; M-1 to L-466; M-1 to V-465; M-1 to L-464; M-1 to A-463; M-1 to L-462; M-1 to G-461; M-1 to L-460; M-1 to P-459; M-1 to T-458; M-1 to L-457; M-1 to S-456; M-1 to C-455; M-1 to T-454; M-1 to L-453; M-1 to S-452; M-1 to P-451; M-1 to L-450; M-1 to A-449; M-1 to G-448; M-1 to S-447; M-1 to G-446; M-1 to E-445; M-1 to S-444; M-1 to D-443; M-1 to G-442; M-1 to T-441; M-1 to G-440; M-1 to G-439; M-1 to G-438; M-1 to G-437; M-1 to S-436; M-1 to G-435; M-1 to A-434; M-1 to Q-433; M-1 to G-432; M-1 to L-431; M-1 to R-430; M-1 to C-429; M-1 to H-428; M-1 to S-427; M-1 to R-426; M-1 to T-425; M-1 to R-424; M-1 to N-423; M-1 to K-422; M-1 to R-421; M-1 to S-420; M-1 to C-419; M-1 to G-418; M-1 to P-417; M-1 to R-416; M-1 to R-415; M-1 to R-414; M-1 to P-413; M-1 to G-412; M-1 to S-411; M-1 to T-410; M-1 to P-409; M-1 to F-408; M-1 to R-407; M-1 to T-406; M-1 to A-405; M-1 to R-404; M-1 to L-403; M-1 to G-402; M-1 to R-401; M-1 to A-400; M-1 to A-399; M-1 to S-398; M-1 to C-397; M-1 to H-396; M-1 to A-395; M-1 to P-394; M-1 to P-393; M-1 to E-392; M-1 to A-391; M-1 to S-390; M-1 to G-389; M-1 to P-388; M-1 to L-387; M-1 to T-386; M-1 to G-385; M-1 to F-384; M-1 to P-383; M-1 to S-382; M-1 to D-381; M-1 to N-380; M-1 to I-379; M-1 to H-378; M-1 to R-377; M-1 to P-376; M-1 to G-375; M-1 to S-374; M-1 to G-373; M-1 to N-372; M-1 to G-371; M-1 to P-370; M-1 to P-369; M-1 to S-368; M-1 to D-367; M-1 to G-366; M-1 to P-365; M-1 to P-364; M-1 to V-363; M-1 to R-362; M-1 to G-361; M-1 to K-360; M-1 to L-359; M-1 to A-358; M-1 to N-357; M-1 to G-356; M-1 to A-355; M-1 to S-354; M-1 to A-353; M-1 to P-352; M-1 to R-351; M-1 to G-350; M-1 to P-349; M-1 to E-348; M-1 to L-347; M-1 to V-346; M-1 to S-345; M-1 to A-344; M-1 to K-343; M-1 to D-342; M-1 to A-341; M-1 to A-340; M-1 to D-339; M-1 to P-338; M-1 to Q-337; M-1 to C-336; M-1 to C-335; M-1 to K-334; M-1 to P-333; M-1 to L-332; M-1 to G-331; M-1 to L-330; M-1 to P-329; M-1 to E-328; M-1 to E-327; M-1 to D-326; M-1 to T-325; M-1 to A-324; M-1 to R-323; M-1 to G-322; M-1 to T-321; M-1 to W-320; M-1 to I-319; M-1 to P-318; M-1 to H-317; M-1 to Y-316; M-1 to P-315; M-1 to G-314; M-1 to T-313; M-1 to A-312; M-1 to V-311; M-1 to A-310; M-1 to C-309; M-1 to G-308; M-1 to Q-307; M-1 to L-306; M-1 to D-305; M-1 to N-304; M-1 to A-303; M-1 to A-302; M-1 to L-301; M-1 to R-300; M-1 to K-299; M-1 to L-298; M-1 to D-297; M-1 to R-296; M-1 to G-295; M-1 to A-294; M-1 to L-293; M-1 to R-292; M-1 to Q-291; M-1 to P-290; M-1 to L-289; M-1 to S-288; M-1 to C-287; M-1 to P-286; M-1 to V-285; M-1 to E-284; M-1 to S-283; M-1 to S-282; M-1 to S-281; M-1 to G-280; M-1 to R-279; M-1 to F-278; M-1 to K-277; M-1 to Q-276; M-1 to L-275; M-1 to W-274; M-1 to A-273; M-1 to W-272; M-1 to L-271; M-1 to P-270; M-1 to R-269; M-1 to A-268; M-1 to R-267; M-1 to C-266; M-1 to D-265; M-1 to C-264; M-1 to V-263; M-1 to W-262; M-1 to P-261; M-1 to N-260; M-1 to D-259; M-1 to N-258; M-1 to L-257; M-1 to R-256; M-1 to L-255; M-1 to Y-254; M-1 to Q-253; M-1 to L-252; M-1 to A-251; M-1 to R-250; M-1 to L-249; M-1 to P-248; M-1 to A-247; M-1 to L-246; M-1 to A-245; M-1 to E-244; M-1 to T-243; M-1 to P-242; M-1 to L-241; M-1 to A-240; M-1 to S-239; M-1 to L-238; M-1 to N-237; M-1 to N-236; M-1 to A-235; M-1 to F-234; M-1 to L-233; M-1 to Y-232; M-1 to L-231; M-1 to T-230; M-1 to M-229; M-1 to L-228; M-1 to R-227; M-1 to G-226; M-1 to L-225; M-1 to D-224; M-1 to R-223; M-1 to F-222; M-1 to A-221; M-1 to H-220; M-1 to P-219; M-1 to H-218; M-1 to V-217; M-1 to H-216; M-1 to A-215; M-1 to V-214; M-1 to R-213; M-1 to N-212; M-1 to Q-211; M-1 to H-210; M-1 to L-209; M-1 to L-208; M-1 to L-207; M-1 to R-206; M-1 to D-205; M-1 to L-204; M-1 to S-203; M-1 to H-202; M-1 to L-201; M-1 to G-200; M-1 to R-199; M-1 to F-198; M-1 to A-197; M-1 to R-196; M-1 to E-195; M-1 to P-194; M-1 to V-193; M-1 to S-192; M-1 to S-191; M-1 to I-190; M-1 to R-189; M-1 to N-188; M-1 to G-187; M-1 to H-186; M-1 to L-185; M-1 to F-184; M-1 to L-183; M-1 to H-182; M-1 to T-181; M-1 to L-180; M-1 to N-179; M-1 to G-178; M-1 to L-177; M-1 to D-176; M-1 to R-175; M-1 to F-174; M-1 to T-173; M-1 to D-172; M-1 to D-171; M-1 to P-170; M-1 to L-169; M-1 to A-168; M-1 to Q-167; M-1 to L-166; M-1 to A-165; M-1 to N-164; M-1 to D-163; M-1 to Q-162; M-1 to L-161; M-1 to Y-160; M-1 to L-159; M-1 to Y-158; M-1 to Q-157; M-1 to L-156; M-1 to A-155; M-1 to A-154; M-1 to L-153; M-1 to G-152; M-1 to R-151; M-1 to F-150; M-1 to L-149; M-1 to G-148; M-1 to P-147; M-1 to G-146; M-1 to L-145; M-1 to E-144; M-1 to Q-143; M-1 to L-142; M-1 to G-141; M-1 to C-140; M-1 to R-139; M-1 to D-138; M-1 to L-137; M-1 to H-136; M-1 to L-135; M-1 to T-134; M-1 to H-133; M-1 to L-132; M-1 to R-131; M-1 to G-130; M-1 to L-129; M-1 to G-128; M-1 to H-127; M-1 to F-126; M-1 to T-125; M-1 to A-124; M-1 to P-123; M-1 to D-122; M-1 to V-121; M-1 to S-120; M-1 to R-119; M-1 to L-118; M-1 to Q-117; M-1 to A-116; M-1 to N-115; M-1 to D-114; M-1 to S-113; M-1 to L-112; M-1 to D-111; M-1 to L-110; M-1 to Q-109; M-1 to E-108; M-1 to L-107; M-1 to L-106; M-1 to A-105; M-1 to L-104; M-1 to G-103; M-1 to T-102; M-1 to F-101; M-1 to A-100; M-1 to A-99; M-1 to A-98; M-1 to D-97; M-1 to I-96; M-1 to R-95; M-1 to A-94; M-1 to L-93; M-1 to V-92; M-1 to N-91; M-1 to S-90; M-1 to H-89; M-1 to L-88; M-1 to W-87; M-1 to L-86; M-1 to I-85; M-1 to T-84; M-1 to L-83; M-1 to N-82; M-1 to R-81; M-1 to C-80; M-1 to A-79; M-1 to R-78; M-1 to F-77; M-1 to S-76; M-1 to A-75; M-1 to A-74; M-1 to P-73; M-1 to V-72; M-1 to H-71; M-1 to S-70; M-1 to I-69; M-1 to R-68; M-1 to N-67; M-1 to G-66; M-1 to H-65; M-1 to L-64; M-1 to F-63; M-1 to I-62; M-1 to R-61; M-1 to Q-60; M-1 to S-59; M-1 to A-58; M-1 to A-57; M-1 to P-56; M-1 to I-55; M-1 to G-54; M-1 to V-53; M-1 to P-52; M-1 to V-51; M-1 to A-50; M-1 to Q-49; M-1 to L-48; M-1 to G-47; M-1 to Q-46; M-1 to Q-45; M-1 to P-44; M-1 to C-43; M-1 to S-42; M-1 to T-41; M-1 to T-40; M-1 to V-39; M-1 to K-38; M-1 to P-37; M-1 to E-36; M-1 to N-35; M-1 to Y-34; M-1 to C-33; M-1 to V-32; M-1 to C-31; M-1 to A-30; M-1 to G-29; M-1 to P-28; M-1 to C-27; M-1 to P-26; M-1 to A-25; M-1 to A-24; M-1 to V-23; M-1 to Q-22; M-1 to W-21; M-1 to A-20; M-1 to Q-19; M-1 to L-18; M-1 to W-17; M-1 to L-16; M-1 to V-15; M-1 to W-14; M-1 to A-13; M-1 to L-12; M-1 to L-11; M-1 to R-10; M-1 to S-9; M-1 to G-8; M-1 to G-7; and M-1 to A-6 of SEQ ID NO:135. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In addition, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides comprising, or alternatively consisting of, one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of SEQ ID NO:135, where n and m are integers as described above. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: M-1 to V-15; K-2 to L-16; R-3 to W-17; A-4 to L-18; S-5 to Q-19; A-6 to A-20; G-7 to W-21; G-8 to Q-22; S-9 to V-23; R-10 to A-24; L-11 to A-25; L-12 to P-26; A-13 to C-27; W-14 to P-28; V-15 to G-29; L-16 to A-30; W-17 to C-31; L-18 to V-32; Q-19 to C-33; A-20 to Y-34; W-21 to N-35; Q-22 to E-36; V-23 to P-37; A-24 to K-38; A-25 to V-39; P-26 to T-40; C-27 to T-41; P-28 to S-42; G-29 to C-43; A-30 to P-44; C-31 to Q-45; V-32 to Q-46; C-33 to G-47; Y-34 to L-48; N-35 to Q-49; E-36 to A-50; P-37 to V-51; K-38 to P-52; V-39 to V-53; T-40 to G-54; T-41 to I-55; S-42 to P-56; C-43 to A-57; P-44 to A-58; Q-45 to S-59; Q-46 to Q-60; G-47 to R-61; L-48 to I-62; Q-49 to F-63; A-50 to L-64; V-51 to H-65; P-52 to G-66; V-53 to N-67; G-54 to R-68; 1-55 to I-69; P-56 to S-70; A-57 to H-71; A-58 to V-72; S-59 to P-73; Q-60 to A-74; R-61 to A-75; 1-62 to S-76; F-63 to F-77; L-64 to R-78; H-65 to A-79; G-66 to C-80; N-67 to R-81; R-68 to N-82; 1-69 to L-83; S-70 to T-84; H-71 to I-85; V-72 to L-86; P-73 to W-87; A-74 to L-88; A-75 to H-89; S-76 to S-90; F-77 to N-91; R-78 to V-92; A-79 to L-93; C-80 to A-94; R-81 to R-95; N-82 to I-96; L-83 to D-97; T-84 to A-98; 1-85 to A-99; L-86 to A-100; W-87 to F-101; L-88 to T-102; H-89 to G-103; S-90 to L-104; N-91 to A-105; V-92 to L-106; L-93 to L-107; A-94 to E-108; R-95 to Q-109; I-96 to L-110; D-97 to D-111; A-98 to L-112; A-99 to S-113; A-100 to D-114; F-101 to N-115; T-102 to A-116; G-103 to Q-117; L-104 to L-118; A-105 to R-119; L-106 to S-120; L-107 to V-121; E-108 to D-122; Q-109 to P-123; L-110 to A-124; D-111 to T-125; L-112 to F-126; S-113 to H-127; D-114 to G-128; N-115 to L-129; A-116 to G-130; Q-117 to R-131; L-118 to L-132; R-119 to H-133; S-120 to T-134; V-121 to L-135; D-122 to H-136; P-123 to L-137; A-124 to D-138; T-125 to R-139; F-126 to C-140; H-127 to G-141; G-128 to L-142; L-129 to Q-143; G-130 to E-144; R-131 to L-145; L-132 to G-146; H-133 to P-147; T-134 to G-148; L-135 to L-149; H-136 to F-150; L-137 to R-151; D-138 to G-152; R-139 to L-153; C-140 to A-154; G-141 to A-155; L-142 to L-156; Q-143 to Q-157; E-144 to Y-158; L-145 to L-159; G-146 to Y-160; P-147 to L-161; G-148 to Q-162; L-149 to D-163; F-150 to N-164; R-151 to A-165; G-152 to L-166; L-153 to Q-167; A-154 to A-168; A-155 to L-169; L-156 to P-170; Q-157 to D-171; Y-158 to D-172; L-159 to T-173; Y-160 to F-174; L-161 to R-175; Q-162 to D-176; D-163 to L-177; N-164 to G-178; A-165 to N-179; L-166 to L-180; Q-167 to T-181; A-168 to H-182; L-169 to L-183; P-170 to F-184; D-171 to L-185; D-172 to H-186; T-173 to G-187; F-174 to N-188; R-175 to R-189; D-176 to I-190; L-177 to S-191; G-178 to S-192; N-179 to V-193; L-180 to P-194; T-181 to E-195; H-182 to R-196; L-183 to A-197; F-184 to F-198; L-185 to R-199; H-186 to G-200; G-187 to L-201; N-188 to H-202; R-189 to S-203; 1-190 to L-204; S-191 to D-205; S-192 to R-206; V-193 to L-207; P-194 to L-208; E-195 to L-209; R-196 to H-210; A-197 to Q-211; F-198 to N-212; R-199 to R-213; G-200 to V-214; L-201 to A-215; H-202 to H-216; S-203 to V-217; L-204 to H-218; D-205 to P-219; R-206 to H-220; L-207 to A-221; L-208 to F-222; L-209 to R-223; H-210 to D-224; Q-211 to L-225; N-212 to G-226; R-213 to R-227; V-214 to L-228; A-215 to M-229; H-216 to T-230; V-217 to L-231; H-218 to Y-232; P-219 to L-233; H-220 to F-234; A-221 to A-235; F-222 to N-236; R-223 to N-237; D-224 to L-238; L-225 to S-239; G-226 to A-240; R-227 to L-241; L-228 to P-242; M-229 to T-243; T-230 to E-244; L-231 to A-245; Y-232 to L-246; L-233 to A-247; F-234 to P-248; A-235 to L-249; N-236 to R-250; N-237 to A-251; L-238 to L-252; S-239 to Q-253; A-240 to Y-254; L-241 to L-255; P-242 to R-256; T-243 to L-257; E-244 to N-258; A-245 to D-259; L-246 to N-260; A-247 to P-261; P-248 to W-262; L-249 to V-263; R-250 to C-264; A-251 to D-265; L-252 to C-266; Q-253 to R-267; Y-254 to A-268; L-255 to R-269; R-256 to P-270; L-257 to L-271; N-258 to W-272; D-259 to A-273; N-260 to W-274; P-261 to L-275; W-262 to Q-276; V-263 to K-277; C-264 to F-278; D-265 to R-279; C-266 to G-280; R-267 to S-281; A-268 to S-282; R-269 to S-283; P-270 to E-284; L-271 to V-285; W-272 to P-286; A-273 to C-287; W-274 to S-288; L-275 to L-289; Q-276 to P-290; K-277 to Q-291; F-278 to R-292; R-279 to L-293; G-280 to A-294; S-281 to G-295; S-282 to R-296; S-283 to D-297; E-284 to L-298; V-285 to K-299; P-286 to R-300; C-287 to L-301; S-288 to A-302; L-289 to A-303; P-290 to N-304; Q-291 to D-305; R-292 to L-306; L-293 to Q-307; A-294 to G-308; G-295 to C-309; R-296 to A-310; D-297 to V-311; L-298 to A-312; K-299 to T-313; R-300 to G-314; L-301 to P-315; A-302 to Y-316; A-303 to H-317; N-304 to P-318; D-305 to I-319; L-306 to W-320; Q-307 to T-321; G-308 to G-322; C-309 to R-323; A-310 to A-324; V-311 to T-325; A-312 to D-326; T-313 to E-327; G-314 to E-328; P-315 to P-329; Y-316 to L-330; H-317 to G-331; P-318 to L-332; 1-319 to P-333; W-320 to K-334; T-321 to C-335; G-322 to C-336; R-323 to Q-337; A-324 to P-338; T-325 to D-339; D-326 to A-340; E-327 to A-341; E-328 to D-342; P-329 to K-343; L-330 to A-344; G-331 to S-345; L-332 to V-346; P-333 to L-347; K-334 to E-348; C-335 to P-349; C-336 to G-350; Q-337 to R-351; P-338 to P-352; D-339 to A-353; A-340 to S-354; A-341 to A-355; D-342 to G-356; K-343 to N-357; A-344 to A-358; S-345 to L-359; V-346 to K-360; L-347 to G-361; E-348 to R-362; P-349 to V-363; G-350 to P-364; R-351 to P-365; P-352 to G-366; A-353 to D-367; S-354 to S-368; A-355 to P-369; G-356 to P-370; N-357 to G-371; A-358 to N-372; L-359 to G-373; K-360 to S-374; G-361 to G-375; R-362 to P-376; V-363 to R-377; P-364 to H-378; P-365 to I-379; G-366 to N-380; D-367 to D-381; S-368 to S-382; P-369 to P-383; P-370 to F-384; G-371 to G-385; N-372 to T-386; G-373 to L-387; S-374 to P-388; G-375 to G-389; P-376 to S-390; R-377 to A-391; H-378 to E-392; 1-379 to P-393; N-380 to P-394; D-381 to A-395; S-382 to H-396; P-383 to C-397; F-384 to S-398; G-385 to A-399; T-386 to A-400; L-387 to R-401; P-388 to G-402; G-389 to L-403; S-390 to R-404; A-391 to A-405; E-392 to T-406; P-393 to R-407; P-394 to F-408; A-395 to P-409; H-396 to T-410; C-397 to S-411; S-398 to G-412; A-399 to P-413; A-400 to R-414; R-401 to R-415; G-402 to R-416; L-403 to P-417; R-404 to G-418; A-405 to C-419; T-406 to S-420; R-407 to R-421; F-408 to K-422; P-409 to N-423; T-410 to R-424; S-411 to T-425; G-412 to R-426; P-413 to S-427; R-414 to H-428; R-415 to C-429; R-416 to R-430; P-417 to L-431; G-418 to G-432; C-419 to Q-433; S-420 to A-434; R-421 to G-435; K-422 to S-436; N-423 to G-437; R-424 to G-438; T-425 to G-439; R-426 to G-440; S-427 to T-441; H-428 to G-442; C-429 to D-443; R-430 to S-444; L-431 to E-445; G-432 to G-446; Q-433 to S-447; A-434 to G-448; G-435 to A-449; S-436 to L-450; G-437 to P-451; G-438 to S-452; G-439 to L-453; G-440 to T-454; T-441 to C-455; G-442 to S-456; D-443 to L-457; S-444 to T-458; E-445 to P-459; G-446 to L-460; S-447 to G-461; G-448 to L-462; A-449 to A-463; L-450 to L-464; P-451 to V-465; S-452 to L-466; L-453 to W-467; T-454 to T-468; C-455 to V-469; S-456 to L-470; L-457 to G-471; T-458 to P-472; and P-459 to C-473 of SEQ ID NO:135. Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The present invention is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein as m-n. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N and C terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also included are polynucleotide sequences encoding a polypeptide consisting of a portion of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 209782, where this portion excludes any integer of amino acid residues from 1 to about 467 amino acids from the amino terminus of the complete amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 209782, or any integer of amino acid residues from 6 to about 473 amino acids from the carboxy terminus, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209782. Polypeptides encoded by these polynucleotides also are encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The translation product of this gene shares sequence homology with ALS (Acid Labile Subunit of Insulin-Like Growth Factor) which is thought to be important in the regulation of IGF availability. As such, it is likely that the product of this gene is involved in the regulation of various proliferation-dependent cellular processes that may be attributable to cancer progression (See Genbank Accession No. gi|184808; all references available through this accession are hereby incorporated by reference herein).

The gene encoding the disclosed cDNA is believed to reside on chromosome 22. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 22.

As described herein or otherwise known in the art, the polynucleotides of the invention have uses that include, but are not limited to, serving as probes or primers in chromosome identification, chromosome mapping, and linkage analysis.

Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, neurodegenerative diseases, growth deficiencies, osteoporosis, catabolic disorders, diabetes, ovarian cancer, neuronal injury spinal injury, post-spinal trauma neurite outgrowth, inflammation, and neuronal injury, and disorders of the central and peripheral nervous system. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the nervous system and other peripheral tissues, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., neural, neuronal, proliferating, and/or other tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution cerebellum and homology to ALS (Acid Labile Subunit of Insulin-Like Growth Factor) indicates that polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of a variety of metabolic disorders, growth deficiencies, osteoporosis, catabolic disorders (including AIDS) and diabetes. Nearly all of the insulin-like growth factor (IGF) in the circulation is bound in a heterotrimeric complex composed of IGF, IGF-binding protein-3, and the acid-labile subunit (ALS). The protein product of this gene therefore may afford the ability to potentiate the biological actions of IGF or similar growth factors and cytokines Studies which demonstrate the beneficial effect of IGF-I in amyotrophic lateral-sclerosis, would suggest a role in this disease as well. Alternatively, the tissue distribution in cancerous ovarian tissue indicates polynucleotides and polypeptides corresponding to this gene are useful for the treatment, diagnosis, and/or prevention of various skin disorders. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, are used to treat, prevent, and/or diagnose diseases and disorders of the nervous system.

In specific embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to diagnose, prognose or monitor neurological diseases, neural injury, and/or spinal cord injury. In other specific embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to treat, prevent, or ameliorate neurological disease, neural injury, and/or spinal cord injury.

In other preferred embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides agonists and antagonists, may be used to diagnose, prognose or monitor diseases and disorders associated with aberrant neurite outgrowth.

By “agonist,” is meant any substance that enhances the function of the polynucleotides or polypeptides of the invention. Classes of molecules that can function as agonists include, but are not limited to, small molecules, antibodies (including fragments or variants thereof, such as Fab fragments, Fab′2 fragments and scFvs), and peptidomimetics. By “antagonist,” is meant any substance that diminishes or abolishes the function of the polynucleotides or polypeptides of the invention. Classes of molecules that can function as antagonists include, but are not limited to, small molecules, antibodies (including fragments or variants thereof, such as Fab fragments, Fab′2 fragments and scFvs) antisense polynucleotides, ribozymes, and peptidomimetics.

In another embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or antagonists thereof (especially neutralizing or antagonistic antibodies) may be used to treat, prevent, and/or ameliorate neurological disease, neural injury, and/or spinal cord injury. Additionally, in other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or anatgonists thereof (especially neutralizing or antagonistic antibodies) may be used to treat, prevent, or ameliorate conditions associated with ameliorate neurological disease, neural injury, and/or spinal cord injury, including, but not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations, and other neurolgical diseases and disorders as described in the “Neural Activity and Neurological Activity diseases” section below,

In a preferred embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or antagonists thereof (especially neutralizing or antagonistic antibodies) may be used to treat, prevent, and/or ameliorate neural injury, and/or spinal cord injury following spinal cord and neural trauma. For example, neural outgrowth and inflammation. Additionally, in other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or anatgonists thereof (especially neutralizing or antagonistic antibodies) may be used to treat, prevent, or ameliorate conditions associated with ameliorate neurological disease, neural injury, and/or spinal cord injury, including, but not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, amylotrophic lateral sclerosis and the like, as well as spinocerebellar degenerations, and other neurolgical diseases and disorders as described in the “Neural Activity and Neurological Activity diseases” section below.

In another embodiment, compositions of the invention i.e., Therapeutics (comprising polynucleotides, polypeptides of the invention, agonists and/or antagonists thereof (including antibodies) as well as fragments and variants of the polynucleotides, polypeptides of the invention, agonists and/or antagonists of the invention) are used in combination with antiinflammatory drugs (e.g., as described herein) and/or drugs used to treat spinal cord injury, neural injury, or neurological disease.

In certain embodiments, the Therapeutics of the invention are administered in combination with agents used to treat psychiatric disorders. Psychiatric drugs that may be administered with the Therapeutics of the invention include, but are not limited to, antipsychotic agents (e.g., chlorpromazine, chlorprothixene, clozapine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, quetiapine, risperidone, thioridazine, thiothixene, trifluoperazine, and triflupromazine), antimanic agents (e.g., carbamazepine, divalproex sodium, lithium carbonate, and lithium citrate), antidepressants (e.g., amitriptyline, amoxapine, bupropion, citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, sertraline, tranylcypromine, trazodone, trimipramine, and venlafaxine), antianxiety agents (e.g., alprazolam, buspirone, chlordiazepoxide, clorazepate, diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulants (e.g., d-amphetamine, methylphenidate, and pemoline).

In other embodiments, the Therapeutics of the invention are administered in combination with agents used to treat neurological disorders. Neurological agents that may be administered with the Therapeutics of the invention include, but are not limited to, antiepileptic agents (e.g., carbamazepine, clonazepam, ethosuximide, phenobarbital, phenyloin, primidone, valproic acid, divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonian agents (e.g., levodopa/carbidopa, selegiline, amantidine, bromocriptine, pergolide, ropinirole, pramipexole, benztropine; biperiden; ethopropazine; procyclidine; trihexyphenidyl, tolcapone), and ALS therapeutics (e.g. riluzole).

The polynucleotides, polypeptides and agonists or antagonists of the invention may also be used for the diagnosis and/or treatment of diseases, disorders, damage or injury of the brain and/or nervous system. Nervous system disorders that can be treated with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the methods of the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to, degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including, but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include nerve compression syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve compression syndrome, neuralgia such as causalgia, cervico-brachial neuralgia, facial neuralgia and trigeminal neuralgia, neuritis such as experimental allergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and radiculities such as polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies which include Congenital Analgesia and Familial Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating and Tetany).

Additional diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include, but are not limited to neoplastic disease of the CNS e.g. glioma, glioblastoma, medulloblastoma, craniopharyngioma, ependyoma, pinealoma, haemangioblastoma, acoustic neuroma, oligodendroglioma, menagioma, neuroblastoma or retinoblastoma and degenerative nerve diseases e.g. Alzheimer's and Parkinson's diseases. Representative uses are described in the “Regeneration” and “Hyperproliferative Disorders” sections below, in Examples 28, 30, 31, 32, 40, 42, and 46, and elsewhere herein. Therapeutics can be used to treat or prevent hyperproliferative or benign dysproliferative disorders e.g. psoriasis and tissue hypertrophy. Ribozymes or antisense nucleic acids can be used to inhibit production of the polypeptides of the invention, to induce regeneration of neurons or to promote structural plasticity of the CNS in disorders where neurite growth, regeneration or maintenance are deficient or desired. The animal models can be used in diagnostic and screening methods for predisposition to disorders and to screen for or test molecules which can treat or prevent disorders or diseases of the CNS.

Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Features of Protein Encoded by Gene No: 21

This gene is expressed primarily in bone marrow, CD34 positive cells, and immune cells, including, neutrophils, T-cells, B-cells, macrophages, monocytes, and dendritic cells and to a lesser extent in brain and tonsils.

In one embodiments, polypeptides comprising the amino acid sequence of the open reading frame upstream of the predicted signal peptide are contemplated by the present invention. Specifically, polypeptides of the invention may comprise, or alternatively consist of the following amino acid sequence: VDGIDKLDIEFLQQFLETHSRGPRLHSPGHASQEATPGANMSSGTELLWPGAALLVLLGVA ASLCVRCSRPGAKRSEKIYQQRSLREDQQSFTGSRTYSLVGQAWPGPLADMAPTRKDKLL QFYPSLEDPASSRYQNFSKGSRHGSEEAYIDPIAMEYYNWGRFSKPPEDDDANSYENVLIC KQKTTETGAQQEGIGGLCRGDLSLSLALKTGPTSGLCPSASPEEDEGI (SEQ ID NO:163). Polynucleotides encoding these polypeptides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Preferred polypeptide fragments may comprise or alternatively consist of one, two, three, four or more of the following amino acid sequence: ASSRYQNFSKGSRHGSEEAYIDPIA (SEQ ID NO:164), MEYYNWGRFSKPPEDDDANSY (SEQ ID NO:165), ENVLICKQKTTETGAQQEGIGGLCRGD (SEQ ID NO:166), VRCSR PGAKRSEKIYQQRSLREDQQSFTGSRTYSLVGQAWPGPLADMAPTRKDKLLQFYPSLEDPA SS (SEQ ID NO:167) and LSLSLALKTGPTSGLCPSASPEEDEGI (SEQ ID NO:168). Polynucleotides encoding these polypeptide fragments (SEQ ID NOs: 164, 165, 166, 167, and/or 168), polynucleotides that hybridize to the complementary strand of these polynucleotides (e.g., under the hybridization conditions described herein) are encompassed by the invention, as are the polypeptides encoded by these hybridizing polynucleotides. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Preferred polypeptides of the present invention comprise, or alternatively consist of one, two, three, four, five, or more of the immunogenic epitopes shown in SEQ ID NO:160 as residues: Ser-29 to Thr-57, Pro-74 to Lys-79, Pro-85 to Glu-107, Tyr-118 to Tyr-136, Gln-144 to Gln-152, Ala-182 to Glu-188. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also preferred are polypeptides comprising the mature polypeptide which is predicted to consist of residues 26-190 of the foregoing sequence (SEQ ID NO:160), and biologically active fragments of the mature polypeptide (e.g., fragments that stimulate the proliferation of bone marrow CD34+ cells).

FIGS. 9A-B show the nucleotide (SEQ ID NO:159) and deduced amino acid sequence (SEQ ID NO:160) of this polypeptide.

FIG. 10 shows an analysis of the amino acid sequence (SEQ ID NO:160). Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, and all were generated using the default settings. In the “Antigenic Index or Jameson Wolf” graph, the positive peaks indicate locations of the highly antigenic regions of the protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained. The domains defined by these graphs are contemplated by the present invention.

The data presented in FIG. 10 are also represented in tabular form in Table 12. The columns are labeled with the headings “Res”, “Position”, and Roman Numerals I XIV. The column headings refer to the following features of the amino acid sequence presented in FIG. 10, and Table 12: “Res”: amino acid residue of SEQ ID NO:160 and FIGS. 9A-B; “Position”: position of the corresponding residue within SEQ ID NO:160 and FIGS. 9A-B; I: Alpha, Regions Garnier Robson; II: Alpha, Regions Chou Fasman; III: Beta, Regions Garnier Robson; IV: Beta, Regions Chou Fasman; V: Turn, Regions Garnier Robson; VI: Turn, Regions Chou Fasman; VII: Coil, Regions Garnier Robson; VIII: Hydrophilicity Plot Kyte Doolittle; IX: Hydrophobicity Plot Hopp Woods; X: Alpha, Amphipathic Regions Eisenberg; XI: Beta, Amphipathic Regions Eisenberg; XII: Flexible Regions Karplus Schulz; XIII: Antigenic Index Jameson Wolf; and XIV: Surface Probability Plot Emini.

Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions. The data representing the structural or functional attributes of the protein set forth in FIG. 10 and/or Table 12, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 12 can be used to determine regions of the protein which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIG. 10, but may, as shown in Table 12, be represented or identified by using tabular representations of the data presented in FIG. 10. The DNA*STAR computer algorithm used to generate FIG. 10 (set on the original default parameters) was used to present the data in FIG. 10 in a tabular format (See Table 12). The tabular format of the data in FIG. 10 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out in FIG. 10 and in Table 12 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in FIGS. 9A-B (SEQ ID NO:160). As set out in FIG. 10 and in Table 12, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO:159 is intended DNA fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:159. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:159. In this context “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, and from about 501 to about 550, and from about 551 to about 600, and from about 601 to about 650, and from about 651 to about 700, and from about 701 to about 750, and from about 751 to about 800, and from about 801 to about 850, and from about 851 to about 900, and from about 901 to about 950, and from about 951 to about 1000, and from about 1001 to about 1050, and from about and from about 1051 to about 1100, and from about 1101 to about 1150, and from about 1151 to about 1200, and from about 1201 to about 1250, and from about 1251 to about 1300, and from about 1301 to about 1350, and from about 1351 to about 1400, and from about 1401 to about 1450, and from about 1451 to about 1500, and from about 1501 to about 1551, and from about 1551 to about 1600, and from about 1601 to about 1650, and from about 1651 to about 1700, and from about 1701 to about 1750, and from about 1751 to about 1797 of SEQ ID NO:159, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. In additional embodiments, the polynucleotides of the invention encode functional attributes of the corresponding protein.

Preferred polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.

Preferably, the polynucleotide fragments of the invention encode a polypeptide which demonstrates a functional activity. By a polypeptide demonstrating a “functional activity” is meant to be a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) or mature-form of the protein. Such functional activities include, but are not limited to, biological activity (e.g., ability to regulate (e.g., stimulate) hematopoiesis in vitro or in vivo), antigenicity, and immunogenicity. The functional activity of polypeptides of the invention, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods described herein.

In addition, assays described herein and otherwise known in the art may routinely be applied to measure biological activity of polypeptides and fragments of the invention, variants derivatives and analogs thereof (e.g., to regulate (e.g., to stimulate or inhibit) hematopoiesis in vitro or in vivo). For example, techniques known in the art (such as for example assaying for thymidine incorporation), may be applied or routinely modified to assay for the ability of the compositions of the invention to inhibit proliferation of hematopoietic cells. Other methods will be known to the skilled artisan and are within the scope of the invention.

Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, etc.) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in FIGS. 9A-B (i.e., SEQ ID NO:159), up to the Glu residue at position number 185 and polynucleotides encoding such polypeptides.

Particularly, N-terminal deletions of the polypeptide can be described by the general formula m-190, where m is an integer from 2 to 184, where m corresponds to the position of the amino acid residue identified in SEQ ID NO:160. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: V-26 to I-190; R-27 to I-190; C-28 to I-190; S-29 to I-190; R-30 to I-190; P-31 to I-190; G-32 to I-190; A-33 to I-190; K-34 to I-190; R-35 to I-190; S-36 to I-190; E-37 to I-190; K-38 to I-190; 1-39 to I-190; Y-40 to I-190; Q-41 to I-190; Q-42 to I-190; R-43 to I-190; S-44 to I-190; L-45 to I-190; R-46 to I-190; E-47 to I-190; D-48 to I-190; Q-49 to I-190; Q-50 to I-190; 5-51 to I-190; F-52 to I-190; T-53 to I-190; G-54 to I-190; S-55 to I-190; R-56 to I-190; T-57 to I-190; Y-58 to I-190; S-59 to I-190; L-60 to I-190; V-61 to I-190; G-62 to I-190; Q-63 to I-190; A-64 to I-190; W-65 to I-190; P-66 to I-190; G-67 to I-190; P-68 to I-190; L-69 to I-190; A-70 to I-190; D-71 to I-190; M-72 to I-190; A-73 to I-190; P-74 to I-190; T-75 to I-190; R-76 to I-190; K-77 to I-190; D-78 to I-190; K-79 to I-190; L-80 to I-190; L-81 to I-190; Q-82 to I-190; F-83 to I-190; Y-84 to I-190; P-85 to I-190; S-86 to I-190; L-87 to I-190; E-88 to I-190; D-89 to I-190; P-90 to I-190; A-91 to I-190; S-92 to I-190; S-93 to I-190; R-94 to I-190; Y-95 to I-190; Q-96 to I-190; N-97 to I-190; F-98 to I-190; S-99 to I-190; K-100 to I-190; G-101 to I-190; S-102 to I-190; R-103 to I-190; H-104 to I-190; G-105 to I-190; S-106 to I-190; E-107 to I-190; E-108 to I-190; A-109 to 1-190; Y-110 to I-190; I-111 to I-190; D-112 to I-190; P-113 to I-190; 1-114 to I-190; A-115 to I-190; M-116 to I-190; E-117 to I-190; Y-118 to I-190; Y-119 to I-190; N-120 to I-190; W-121 to I-190; G-122 to I-190; R-123 to I-190; F-124 to I-190; S-125 to I-190; K-126 to I-190; P-127 to I-190; P-128 to I-190; E-129 to I-190; D-130 to I-190; D-131 to I-190; D-132 to I-190; A-133 to I-190; N-134 to I-190; S-135 to I-190; Y-136 to I-190; E-137 to I-190; N-138 to I-190; V-139 to I-190; L-140 to I-190; 1-141 to I-190; C-142 to I-190; K-143 to I-190; Q-144 to I-190; K-145 to I-190; T-146 to I-190; T-147 to I-190; E-148 to I-190; T-149 to I-190; G-150 to I-190; A-151 to I-190; Q-152 to I-190; Q-153 to I-190; E-154 to I-190; G-155 to I-190; 1-156 to I-190; G-157 to I-190; G-158 to I-190; L-159 to I-190; C-160 to I-190; R-161 to I-190; G-162 to I-190; D-163 to I-190; L-164 to I-190; S-165 to I-190; L-166 to I-190; S-167 to I-190; L-168 to I-190; A-169 to I-190; L-170 to I-190; K-171 to I-190; T-172 to I-190; G-173 to I-190; P-174 to I-190; T-175 to I-190; S-176 to I-190; G-177 to I-190; L-178 to I-190; C-179 to I-190; P-180 to I-190; S-181 to I-190; A-182 to I-190; S-183 to I-190; P-184 to I-190; and E-185 to I-190, of SEQ ID NO:160. Polypeptides encoding these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind ligand) may still be retained. For example the ability of the shortened mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C terminus. Whether a particular polypeptide lacking C terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an mutein with a large number of deleted C terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the polypeptide shown in FIGS. 9A-B (SEQ ID NO:160), as described by the general formula 1-n, where n is an integer from 6 to 184 where n corresponds to the position of amino acid residue identified in SEQ ID NO:160. More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, an amino acid sequence selected from the group: V-26 to G-189; V-26 to E-188; V-26 to D-187; V-26 to E-186; V-26 to E-185; V-26 to P-184; V-26 to S-183; V-26 to A-182; V-26 to S-181; V-26 to P-180; V-26 to C-179; V-26 to L-178; V-26 to G-177; V-26 to S-176; V-26 to T-175; V-26 to P-174; V-26 to G-173; V-26 to T-172; V-26 to K-171; V-26 to L-170; V-26 to A-169; V-26 to L-168; V-26 to S-167; V-26 to L-166; V-26 to S-165; V-26 to L-164; V-26 to D-163; V-26 to G-162; V-26 to R-161; V-26 to C-160; V-26 to L-159; V-26 to G-158; V-26 to G-157; V-26 to I-156; V-26 to G-155; V-26 to E-154; V-26 to Q-153; V-26 to Q-152; V-26 to A-151; V-26 to G-150; V-26 to T-149; V-26 to E-148; V-26 to T-147; V-26 to T-146; V-26 to K-145; V-26 to Q-144; V-26 to K-143; V-26 to C-142; V-26 to I-141; V-26 to L-140; V-26 to V-139; V-26 to N-138; V-26 to E-137; V-26 to Y-136; V-26 to S-135; V-26 to N-134; V-26 to A-133; V-26 to D-132; V-26 to D-131; V-26 to D-130; V-26 to E-129; V-26 to P-128; V-26 to P-127; V-26 to K-126; V-26 to S-125; V-26 to F-124; V-26 to R-123; V-26 to G-122; V-26 to W-121; V-26 to N-120; V-26 to Y-119; V-26 to Y-118; V-26 to E-117; V-26 to M-116; V-26 to A-115; V-26 to I-114; V-26 to P-113; V-26 to D-112; V-26 to I-111; V-26 to Y-110; V-26 to A-109; V-26 to E-108; V-26 to E-107; V-26 to S-106; V-26 to G-105; V-26 to H-104; V-26 to R-103; V-26 to S-102; V-26 to G-101; V-26 to K-100; V-26 to S-99; V-26 to F-98; V-26 to N-97; V-26 to Q-96; V-26 to Y-95; V-26 to R-94; V-26 to S-93; V-26 to S-92; V-26 to A-91; V-26 to P-90; V-26 to D-89; V-26 to E-88; V-26 to L-87; V-26 to S-86; V-26 to P-85; V-26 to Y-84; V-26 to F-83; V-26 to Q-82; V-26 to L-81; V-26 to L-80; V-26 to K-79; V-26 to D-78; V-26 to K-77; V-26 to R-76; V-26 to T-75; V-26 to P-74; V-26 to A-73; V-26 to M-72; V-26 to D-71; V-26 to A-70; V-26 to L-69; V-26 to P-68; V-26 to G-67; V-26 to P-66; V-26 to W-65; V-26 to A-64; V-26 to Q-63; V-26 to G-62; V-26 to V-61; V-26 to L-60; V-26 to S-59; V-26 to Y-58; V-26 to T-57; V-26 to R-56; V-26 to S-55; V-26 to G-54; V-26 to T-53; V-26 to F-52; V-26 to S-51; V-26 to Q-50; V-26 to Q-49; V-26 to D-48; V-26 to E-47; V-26 to R-46; V-26 to L-45; V-26 to S-44; V-26 to R-43; V-26 to Q-42; V-26 to Q-41; V-26 to Y-40; V-26 to I-39; V-26 to K-38; V-26 to E-37; V-26 to S-36; V-26 to R-35; V-26 to K-34; V-26 to A-33; V-26 to G-32; and V-26 to P-31 of SEQ ID NO:160. Polypeptides encoding these polynucleotides are also encompassed by the invention. Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In addition, any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of SEQ ID NO:160, where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also included are a nucleotide sequence encoding a polypeptide consisting of a portion of the complete amino acid sequence encoded by the cDNA clone contained in ATCC™ Deposit No. 209889, where this portion excludes any integer of amino acid residues from 1 to about 184 amino acids from the amino terminus of the complete amino acid sequence encoded by the cDNA clone contained in ATCC™ Deposit No. 209889, or any integer of amino acid residues from 1 to about 190 amino acids from the carboxy terminus, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC™ Deposit No. 209889. Polynucleotides encoding all of the above deletion mutant polypeptide forms also are provided.

The present application is also directed to proteins containing polypeptides at least 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the polypeptide sequence set forth herein m-n. In preferred embodiments, the application is directed to proteins containing polypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N and C terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, disorders affecting the immune and hematopoietic systems, particularly hematopoiesis. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system and hematopoeitic system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

This gene has been found to stimulate the proliferation of bone marrow CD34+ cells. This assay which is described in Example 52 herein is based on the ability of human CD34+ to proliferate in presence of hematopoietic growth factors and evaluates the ability of the polypeptides of the invention, and agonists and antagonists thereof, to stimulate or inhibit this proliferation.

The tissue distribution in immune and hematopoietic cells and tissues and the ability to stimulate the proliferation of bone marrow CD34+ cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51 and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. Moreover, the protein represents a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

The polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof, can also be employed to inhibit the proliferation and differentiation of hematopoietic cells and therefore may be employed to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy. This antiproliferative effect may allow administration of higher doses of chemotherapeutic agents and, therefore, more effective chemotherapeutic treatment.

The polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof, may also be employed for the expansion of immature hematopoietic progenitor cells, for example, granulocytes, macrophages or monocytes, by temporarily preventing their differentiation. These bone marrow cells may be cultured in vitro. Thus, polynucleotides and/or polypeptides of the invention, or agonists or antagonists thereof, may be useful as a modulator of hematopoietic stem cells in vitro for the purpose of bone marrow transplantation and/or gene therapy. Since stem cells are rare and are most useful for introducing genes into for gene therapy, polynucleotides and/or polypeptides of the invention can be used to isolate enriched populations of stem cells. Stem cells can be enriched by culturing cells in the presence of cytotoxins, such as 5-Fu, which kills rapidly dividing cells, where as the stem cells will be protected by polynucleotides and/or polypeptides of the invention. These stem cells can be returned to a bone marrow transplant patient or can then be used for transfection of the desired gene for gene therapy. In addition, this gene can be injected into animals which results in the release of stem cells from the bone marrow of the animal into the peripheral blood. These stem cells can be isolated for the purpose of autologous bone marrow transplantation or manipulation for gene therapy. After the patient has finished chemotherapy or radiation treatment, the isolated stem cells can be returned to the patient.

Polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.

This gene product may also be involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g. by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.

This gene may also have a very wide range of biological activities. Representative uses are described in the “Chemotaxis” and “Binding Activity” sections below, in Examples 31, 42, 44, 45, 46, 47, 49, 50, and 51, and elsewhere herein. Briefly, the protein may possess the following activities: cytokine, cell proliferation/differentiation modulating activity or induction of other cytokines; immunostimulating/immunosuppressant activities (e.g. for treating human immunodeficiency virus infection, cancer, autoimmune diseases and allergy); regulation of hematopoiesis (e.g. for treating anemia or as adjunct to chemotherapy); stimulation or growth of bone, cartilage, tendons, ligaments and/or nerves (e.g. for treating wounds, stimulation of follicle stimulating hormone (for control of fertility); chemotactic and chemokinetic activities (e.g. for treating infections, tumors); hemostatic or thrombolytic activity (e.g. for treating hemophilia, cardiac infarction etc.); anti-inflammatory activity (e.g. for treating septic shock, Crohn's disease); as antimicrobials; for treating psoriasis or other hyperproliferative diseases; for regulation of metabolism, and behavior. Also contemplated is the use of the corresponding nucleic acid in gene therapy procedures. Based upon the proteins immune cell specific message distribution, it may be involved in many aspects of the immune response, especially its initial stages, inflammation, allograft rejection, infectious disease response etc. It is frequently found in the hematopoietic cell cDNA libraries. Thus, this factor could be involved in the control of hematopoietic cell proliferation, differentiation, and function. Based on this one can postulate its use in the management of anemias, leukemias, neutropenia, thrombocytopenia, autoimmune diseases, blood tissue engraftment, and poikilothromerythromatosis. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

The gene encoding the disclosed cDNA is believed to reside on chromosome 7. Accordingly, polynucleotides related to this invention are useful as a marker in linkage analysis for chromosome 7.

Features of Protein Encoded by Gene No: 22

Preferred polypeptides of the invention comprise the following amino acid sequences: FEIALPRESNITVLIKLGTPTLLAKPCYIVISKRHITMLSIKSGERIVFTFSCQSPENHFVIEIQK NIDCMSGPCPFGEVQLQPSTSLLPTLNRTFIWDVKAHKSIGLELQFSIPRLRQIGPGESCPDG VTHSISGRIDATVVRIGTFCSNGTVSRIKM (SEQ ID NO:171), MAGLNCGVSIALLGVLLLGAARLPRGAEAFEIALPRESNITVLIKLGTPTLLAKPCYIVISKR HITMLSIKSGERIVFTFSCQSPENHFVIEIQKNIDCMSGPCPFGEVQLQPSTSLLPTLNRTFIWD VKAHKSIGLELQFSIPRLRQIGPGESCPDGVTHSISGRIDATVVRIGTFCSNGTVSRIKMQEGV KMALHLPWFHPRNVSGFSIANRSSIKRLCIIESVFEGEGSATLMSANYPEGFPEDELMTWQF VVPAHLRASVSFLNFNLSNCERKEERVEYYIPGSTTNPEVFKLEDKQPGNMAGNFNLSLQG CDQDAQSPGILRLQFQVLVQHPQNESNKIYVVDLSNERAMSLTIEPRPVKQSRKFVPGCFV CLESRTCSSNLTLTSGSKHKISFLCDDLTRLWMNVEKP (SEQ ID NO:172) and/or GTRAAPGLGAWGRRSPPSFSPPRPRRPGVMAGLNCGVSIALLGVLLLGAARLPRGAEAFEI ALPRESNITVLIKLGTPTLLAKPCYIVISKRHITMLSIKSGERIVFTFSCQSPENHFVIEIQKNID CMSGPCPFGEVQLQPSTSLLPTLNRTFIWDVKAHKSIGLELQFSIPRLRQIGPGESCPDGVTH SISGRIDATVVRIGTFCSNGTVSRIKMQEGVKMALHLPWFHPRNVSGFSIANRSSIKRLCIIES VFEGEGSATLMSANYPEGFPEDELMTWQFVVPAHLRASVSFLNFNLSNCERKEERVEYYIP GSTTNPEVFKLEDKQPGNMAGNFNLSLQGCDQDAQSPGILRLQFQVLVQHPQNESNKIYV VDLSNERAMSLTIEPRPVKQSRKFVPGCFVCLESRTCSSNLTLTSGSKHKISFLCDDLTRLW MNVEKP (SEQ ID NO:173). Polynucleotides encoding these polypeptides are also provided.

This gene is expressed primarily in placenta, and to a lesser extent in, prostate and ovary.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, male and female infertility, and associated disorders of the reproductive system. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the reproductive system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., reproductive, or cancerous and wounded tissues) or bodily fluids (e.g., lymph, amniotic fluid, seminal fluid, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

The tissue distribution of this gene in the prostate, placenta and ovary indicates that polynucleotides and polypeptides corresponding to this gene are useful for treatment, prevention, and/or diagnosis of male or female infertility, endocrine disorders, fetal deficiencies, ovarian failure, amenorrhea, ovarian cancer, benign prostate hyperplasia and prostate cancer. Similarly, the tissue distribution indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of cancer and other proliferative disorders. Expression within placental tissue and other cellular sources marked by proliferating cells indicates that this protein may play a role in the regulation of cellular division. Similarly, embryonic development also involves decisions involving cell differentiation and/or apoptosis in pattern formation. Thus, this protein may also be involved in apoptosis or tissue differentiation and could again be useful in cancer therapy. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:169 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 2195 of SEQ ID NO:169, b is an integer of 15 to 2209, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:169, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 23

The translation product of this gene was found to be homologous to cell surface adhesion molecule (CAM) proteins. Based on the sequence similarity, the translation product of this gene is expected to share at least some biological activities with CAM proteins. Such activities are known in the art, some of which are described elsewhere herein. A preferred polypeptide variant of the invention comprises the following amino acid sequence: MLCPWRTANLGLLLILTIFLVAEAEGAAQPNNSLMLQTSKENHALASSSLCMDEKQITQNY SKVLAEVNTSWPVKMATNAVLCCPPIALRNLIIITWEIILRGQPSCTKAYKKETNETKETNC TDERITWVSRPDQNSDLQIRTVAITHDGYYRCIMVTPDGNFHRGYHLQVLVTPEVTLFQNR NRTAVCKAVAGKPAAHISWIPEGDCATKQEYWSNGTVTVKSTCHWEVHNVSTVNCHVSH LTGNKSLYIELLPVPGAKKSSKLYIPYIILTIIILTIVGXIWLLKVNGCXKYKLNKPESTPVVEE DEMQPYAFYTEKNNPLXXTTNKVKASEALQSEVDTDLHTL (SEQ ID NO:178). Polynucleotides encoding these polypeptides are also provided.

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 271-287 of the amino acid sequence referenced in Table 1A for this gene. Moreover, a cytoplasmic tail encompassing amino acids 288 to 348 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ia membrane proteins.

This gene is expressed primarily in dendritic cells.

Therefore, polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immunodeficiency, tumor necrosis, infection, lymphomas, auto-immunities, cancer, metastasis, wound healing, inflammation, anemias (leukemia) and other hematopoeitic disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, hematopoietic, and cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder. Preferred polypeptides of the present invention comprise immunogenic epitopes shown in SEQ ID NO:175 as residues: Asp-53 to Tyr-61, Pro-105 to Ile-128, Arg-133 to Leu-140, Gln-182 to Ala-188, Pro-205 to Asn-218, Gly-259 to Ala-264, Asn-290 to Ser-302, Glu-307 to Tyr-314, Tyr-317 to Lys-332. Polynucleotides encoding said polypeptides are also provided.

The tissue distribution in dendritic cells indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and treatment of immune disorders including: leukemias, lymphomas, auto-immunities, immunodeficiencies (e.g. AIDS), immuno-supressive conditions (transplantation) and hematopoeitic disorders. In addition this gene product may be applicable in conditions of general microbial infection, inflammation or cancer. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51, and elsewhere herein. Briefly, the expression of this gene product indicates a role in regulating the proliferation; survival; differentiation; and/or activation of hematopoietic cell lineages, including blood stem cells. This gene product is involved in the regulation of cytokine production, antigen presentation, or other processes suggesting a usefulness in the treatment of cancer (e.g. by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the natural gene product is involved in immune functions. Therefore it is also useful as an agent for immunological disorders including arthritis, asthma, immunodeficiency diseases such as AIDS, leukemia, rheumatoid arthritis, granulomatous disease, inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia, psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity; immune reactions to transplanted organs and tissues, such as host-versus-graft and graft-versus-host diseases, or autoimmunity disorders, such as autoimmune infertility, lense tissue injury, demyelination, systemic lupus erythematosis, drug induced hemolytic anemia, rheumatoid arthritis, Sjogren's disease, and scleroderma.

Moreover, the protein may represent a secreted factor that influences the differentiation or behavior of other blood cells, or that recruits hematopoietic cells to sites of injury. Thus, this gene product is thought to be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:174 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 3784 of SEQ ID NO:174, b is an integer of 15 to 3798, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:174, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 24

The translation product of this gene shares sequence homology with CMRF-35 antigen [Homo sapiens] (See, e.g., Genbank accession number AAD01646 and CAA46948; all references available through these accessions are hereby incorporated by reference herein), which is thought to be important as a cell membrane antigen present on the surface of monocytes, neutrophils, a proportion of peripheral blood T and B lymphocytes and lymphocytic cell lines.

The translation product of this gene also shares sequence homology with PIGR-1 protein (see, e.g., Genseq accession number W99070 which is a member of the Immunoglobulin (Ig) superfamily). All references available through this Genseq accession are hereby incorporated by reference herein. Based on the sequence similarity, the translation product of this clone is expected to share at least some biological activities with proteins of the Immunoglobulin superfamily such as, for example, PIGR-1 and CMRF-35 Ag. Such activities are known in the art, some of which are described elsewhere herein.

In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: EGGSSRARXSTSRRLGVCSLFLLPGSTEGNGDLSEEK (SEQ ID NO:181). Moreover, fragments and variants of these polypeptides (such as, for example, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind polypeptides of the invention are also encompassed by the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The polypeptide of this gene has been determined to have a transmembrane domain at about amino acid position 157-173 of the amino acid sequence referenced in Table 1A for this gene. Moreover, a cytoplasmic tail encompassing amino acids 174-290 of this protein has also been determined. Based upon these characteristics, it is believed that the protein product of this gene shares structural features to type Ia membrane proteins.

This gene is expressed primarily in eosinophils, and to a lesser extent in dendritic cells and activated monocytes.

Polynucleotides and polypeptides of the invention are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of diseases and conditions which include, but are not limited to, immune disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful in providing immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the immune system, expression of this gene at significantly higher or lower levels may be routinely detected in certain tissues or cell types (e.g., immune, cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue or bodily fluid from an individual not having the disorder.

Preferred polypeptides of the present invention comprise, or alternatively consist of one, two, three, or all four, of the immunogenic epitopes shown in SEQ ID NO:180 as residues: Ser-69 to Arg-79, Ile-82 to Arg-89, Pro-129 to Ser-137, Leu-146 to Lys-151. Polynucleotides encoding said polypeptides are also encompassed by the invention.

The tissue distribution in eosinophils, monocytes, and dendritic cells, and the homology to CMRF-35 antigen, indicates that polynucleotides and polypeptides corresponding to this gene are useful for the diagnosis and/or treatment of immune disorders. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51, and elsewhere herein. Expression of this gene product in eosinophils, monocytes, and dendritic cells also strongly indicates a role for this protein in immune function and immune surveillance. This gene product may be involved in the regulation of cytokine production, antigen presentation, or other processes that may also suggest a usefulness in the treatment of cancer (e.g., by boosting immune responses). Since the gene is expressed in cells of lymphoid origin, the gene or protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues. Therefore it may be also used as an agent for immunological disorders including arthritis, asthma, immune deficiency diseases such as AIDS, leukemia, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, sepsis, acne, and psoriasis. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:179 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 1782 of SEQ ID NO:179, b is an integer of 15 to 1796, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:179, and where b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 25

This gene is expressed in the following tissues/cDNA libraries: B Cell lymphoma; pBMC stimulated w/poly I/C; B-cells (unstimulated); NCI_CGAP_GCB1.

The tissue distribution indicates polynucleotides and polypeptides corresponding to this gene, as well as antibodies against those polypeptides, may be useful for the diagnosis, prevention, and/or treatment of immune system disorders; particularly immune cell proliferative disorders (e.g. leukemia), autoimmune disorders, and immunodeficiencies (including immunodeficiencies caused by genetic factors, microbial pathogens (e.g. HIV), chemotherapy, and radiation). See “Immune Activity” section, infra. The tissue distribution also indicates polynucleotides and polypeptides corresponding to this gene, as well as antibodies against those polypeptides, may be useful for the diagnosis, prevention, and/or treatment of cancer and other hyperproliferative disorders (e.g., see “Hyperproliferative Disorders” section, infra)

Features of Protein Encoded by Gene No: 26

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. The polypeptide of the present invention has been putatively identified as a human integrin alpha 11 homolog derived from a human osteoblast II cDNA library. More particularly, the polypeptide of the present invention has been putatively identified as a human integrin alpha 11-subunit homolog, sometimes hereafter referred to as “integrin alpha 11”, “integrin alpha 11-subunit”, “a11”, “A11-subunit”, and/or “Integrin a11-subunit”. The invention also relates to inhibiting the action of such polypeptides. The integrins are a large family of cell adhesion molecules consisting of noncovalently associated ab heterodimers.

We have cloned and sequenced a novel human integrin a-subunit cDNA, designated a11. The a11 cDNA encodes a protein with a 22 amino acid signal peptide, a large 1120 residue extracellular domain that contains an I-domain of 207 residues and is linked by a transmembrane domain to a short cytoplasmic domain of 24 amino acids. The deduced a11 protein shows the typical structural features of integrin a-subunits and is similar to a distinct group of a-subunits from collagen-binding integrins. However, it differs from most integrin a-chains by an incompletetely preserved cytoplasmic GFFKR motif.

The human ITGA11 gene was located to bands q22.3-23 on chromosome 15, and its transcripts were found predominantly in bone, cartilage as well as in cardiac and skeletal muscle. Expression of the 5.5 kilobase all mRNA was also detectable in ovary and small intestine.

All vertebrate cells express members of the integrin family of cell adhesion molecules, which mediate cellular adhesion to other cells and extracellular subtratum, cell migration and participate in important physiologic processes from signal transduction to cell proliferation and differentiation (Hynes, 92; Springer, 92).

Integrins are structurally homologous heterodimeric type-I membrane glycoproteins formed by the noncovalent association of one of eight b-subunits with one of the 17 different a-subunits described to date, resulting in at least 22 different ab complexes. Their binding specificities for cellular and extracellular ligands are determined by both subunits and are dynamically regulated in a cell-type-specific mode by the cellular environment as well as by the developmental and activation state of the cell (Diamond and Springer, 94). In integrin a-subunits, the aminoterminal region of the large extracellular domain consists of a seven-fold repeated structure which is predicted to fold into a b-propeller domain (Corbi et al., 1987; Springer, 1997). The three or four C-terminal repeats contain putative divalent cation binding motifs that are thought to be important for ligand binding and subunit association (Diamond and Springer, 94). The a1, a2, a 10, aD, aE, aL, aM and aX-subunits contain an approximately 200 amino acid I-domain inserted between the second and third repeat that is not present in other a-chains (Larson et al., 1989). Several isolated I-domains have been shown to independently bind the ligands of the parent integrin heterodimer (Kamata and Takada, 1994; Randi and Hogg, 1994). The a3, a5-8, aIIb and aV-subunits are proteolytically processed at a conserved site into disulphide-linked heavy and light chains, while the a4-subunit is cleaved at a more aminoterminal site into two fragments that remain noncovalently associated (Hemler et al., 90). Additional a-subunit variants are generated by alternative splicing of primary transcripts (Ziober et al., 93; Delwel et al., 95; Leung et al., 98).

The extracellular domains of a-integrin subunits are connected by a single spanning transmembrane domain to short, diverse cytoplasmic domains whose only conserved feature is a membrane-proximal KXGFF(K/R)R motif (Sastry and Horwitz, 1993). The cytoplasmic domains have been implicated in the cell-type-specific modulation of integrin affinity states (Williams et al., 1994).

The polypeptide of the present invention has been putatively identified as a member of the integrin family and has been termed integrin alpha 11 subunit (“a11”). This identification has been made as a result of amino acid sequence homology to the human integrin alpha 1 subunit (See Genbank Accession No. gi|346210).

FIGS. 11A-F show the nucleotide (SEQ ID NO:186) and deduced amino acid sequence (SEQ ID NO:187) of a11. Predicted amino acids from about 1 to about 22 constitute the predicted signal peptide (amino acid residues from about 1 to about 22 in SEQ ID NO:187) and are represented by the underlined amino acid regions; amino acids from about 666 to about 682, and/or amino acids from about 1145 to about 1161 constitute the predicted transmembrane domains (amino acids from about 666 to about 682, and/or amino acids from about 1145 to about 1161 in SEQ ID NO:187) and are represented by the double underlined amino acids; and amino acids from about 64 to about 96 constitute the predicted immunoglobulin and major histocompatibility complex protein domain (amino acids from about 64 to about 96 in SEQ ID NO:187) and are represented by the bold amino acids.

FIGS. 12A-E shows the regions of similarity between the amino acid sequences of the integrin alpha 11 subunit (all) protein (SEQ ID NO:187) and the human integrin alpha 1 subunit (SEQ ID NO:191).

FIG. 13 shows an analysis of the integrin alpha 11 subunit (all) amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.

Its translation product has homology to the characteristic immunoglobulin and major histocompatibility complex protein domain of integrin family members. As shown in FIGS. 11A-F, a11 has transmembrane domains (the transmembrane domains comprise amino acids 666-682 and/or 1145-1161 of SEQ ID NO:187; which correspond to amino acids 666-682 and/or 1145-1161 of FIGS. 11A-F) with strong conservation between other members of the integrin family. The polynucleotide contains an open reading frame encoding the all polypeptide of 1189 amino acids. The present invention exhibits a high degree of homology at the amino acid level to the human integrin alpha 1 subunit (as shown in FIGS. 12A-E).

Preferred polypeptides of the invention comprise, or alternatively consist of, the following amino acid sequence: TNGYQKTGDVYKCPVIHGNCTKLNLGRVTLSNV (SEQ ID NO:190). Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (e.g., fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind these fragments and variants of the invention are also encompassed by the invention. Polynucleotides encoding these fragments and variants are also encompassed by the invention.

Preferred polypeptides of the present invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three, or all thirty-three of the immunogenic epitopes shown in SEQ ID NO:187 as residues: Phe-23 to Arg-31, Leu-62 to Asp-72, Val-96 to Asp-101, Thr-111 to Asn-116, Glu-128 to Thr-135, Val-142 to Ser-149, Asn-217 to Val-222, Glu-233 to Arg-241, Gly-272 to Leu-280, Gln-286 to Thr-293, Tyr-303 to Ile-308, Gly-354 to Thr-360, Glu-408 to Lys-419, Glu-508 to Lys-514, Arg-521 to Val-526, Gly-529 to Phe-542, Asp-551 to Tyr-557, Thr-587 to Thr-593, His-656 to Asp-665, Met-697 to Arg-705, Asp-709 to Thr-716, Glu-755 to Gly-760, Asn-779 to His-786, Leu-810 to Asp-816, Leu-844 to Ala-851, Gln-871 to Gly-877, Glu-884 to Gln-889, Ser-931 to Asn-943, Ser-974 to Ile-982, Gly-1039 to Gln-1058, Arg-1121 to Arg-1127, Ser-1134 to Trp-1139, and/or Ser-1172 to Pro-1183. Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (e.g., fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind these fragments and variants of the invention are also encompassed by the invention. Polynucleotides encoding these fragments and variants are also encompassed by the invention.

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the all polypeptide having the amino acid sequence shown in FIGS. 11A-F (SEQ ID NO:187). The nucleotide sequence shown in FIGS. 11A-F (SEQ ID NO:186) was obtained by sequencing a cloned cDNA (HOHBY69), which was deposited on November 17 at the American Type Culture Collection, and given Accession Number 203484.

The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated DNA molecule having the nucleotide sequence of the deposited cDNA or the nucleotide sequence shown in SEQ ID NO:186 is intended DNA fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments 50-1500 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:186. By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:186. In this context “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Representative examples of all polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to about 50, from about 51 to about 100, from about 101 to about 150, from about 151 to about 200, from about 201 to about 250, from about 251 to about 300, from about 301 to about 350, from about 351 to about 400, from about 401 to about 450, from about 451 to about 500, from about 501 to about 550, from about 551 to about 600, from about 601 to about 650, from about 651 to about 700, from about 701 to about 750, from about 751 to about 800, from about 801 to about 850, from about 851 to about 900, from about 901 to about 950, from about 951 to about 1000, from about 1001 to about 1050, from about 1051 to about 1100, from about 1101 to about 1150, from about 1151 to about 1200, from about 1201 to about 1250, from about 1251 to about 1300, from about 1301 to about 1350, from about 1351 to about 1400, from about 1401 to about 1450, from about 1451 to about 1500, from about 1501 to about 1550, from about 1551 to about 1600, from about 1601 to about 1650, from about 1651 to about 1700, from about 1701 to about 1750, from about 1751 to about 1800, from about 1801 to about 1850, from about 1851 to about 1900, from about 1901 to about 1950, from about 1951 to about 2000, from about 2001 to about 2050, from about 2051 to about 2100, from about 2101 to about 2150, from about 2151 to about 2200, from about 2201 to about 2250, from about 2251 to about 2300, from about 2301 to about 2350, from about 2351 to about 2400, from about 2401 to about 2450, from about 2451 to about 2500, from about 2501 to about 2550, from about 2551 to about 2600, from about 2601 to about 2650, from about 2651 to about 2700, from about 2701 to about 2750, from about 2751 to about 2800, from about 2801 to about 2850, from about 2851 to about 2900, from about 2901 to about 2950, from about 2951 to about 3000, from about 3001 to about 3050, from about 3051 to about 3100, from about 3101 to about 3150, from about 3151 to about 3200, from about 3201 to about 3250, from about 3251 to about 3300, from about 3301 to about 3350, from about 3351 to about 3400, from about 3401 to about 3450, from about 3451 to about 3500, from about 3501 to about 3550, from about 3551 to about 3600, from about 3601 to about 3650, from about 3651 to about 3700, from about 3701 to about 3750, from about 3751 to about 3800, from about 3801 to about 3850, from about 3851 to about 3900, from about 3901 to about 3950, from about 3951 to about 4000, from about 4001 to about 4050, from about 4051 to about 4100, from about 4101 to about 4150, from about 4151 to about 4200, from about 4201 to about 4250, from about 4251 to about 4300, from about 4301 to about 4350, from about 4351 to about 4400, from about 4401 to about 4450, from about 4451 to about 4500, from about 4501 to about 4550, from about 4551 to about 4600, from about 4601 to about 4650, from about 4651 to about 4700, from about 4701 to about 4750, from about 4751 to about 4800, from about 4801 to about 4850, from about 4851 to about 4900, from about 4901 to about 4950, from about 4951 to about 4995, from about, from about 1 to about 236, from about 144 to about 188, from about 231 to about 276 of SEQ ID NO:186, or the complementary strand thereto, or the cDNA contained in the deposited gene. In this context “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.

Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of, any one of the transmembrane domains (amino acid residues from about 666 to about 682 and/or 1145 to about 1161 in FIGS. 11A-F (amino acids from about 666 to about 682 and/or 1145 to about 1161 in SEQ ID NO:187), in addition to the immunoglobulin and major histocompatibility complex protein domain (amino acid residues from about 64 to about 96 in FIGS. 11A-F (amino acids from about 64 to about 96 in SEQ ID NO:187). Since the location of these domains have been predicted by computer analysis, one of ordinary skill would appreciate that the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to 15 amino acid residues) depending on the criteria used to define each domain. In additional embodiments, the polynucleotides of the invention encode functional attributes of a11.

Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of the present invention.

The data representing the structural or functional attributes of all set forth in FIG. 13 and/or Table 13, as described above, was generated using the various modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table 13 can be used to determine regions of all which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIG. 13, but may, as shown in Table 13, be represented or identified by using tabular representations of the data presented in FIG. 13. The DNA*STAR computer algorithm used to generate FIG. 13 (set on the original default parameters) was used to present the data in FIG. 13 in a tabular format (See Table 13). The tabular format of the data in FIG. 13 is used to easily determine specific boundaries of a preferred region. The above-mentioned preferred regions set out in FIG. 13 and in Table 13 include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in FIGS. 11A-F. As set out in FIG. 13 and in Table 13, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions, Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenic index and Emini surface-forming regions. Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, etc.) may still be retained. For example, the ability of shortened all muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an all mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six all amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the all amino acid sequence shown in FIGS. 11A-F, up to the threonine residue at position number 1184 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues n1-1189 of FIGS. 11A-F, where n1 is an integer from 2 to 1184 corresponding to the position of the amino acid residue in FIGS. 11A-F (which is identical to the sequence shown as SEQ ID NO:187). In another embodiment, N-terminal deletions of the all polypeptide can be described by the general formula n2-1189, where n2 is a number from 2 to 1184, corresponding to the position of amino acid identified in FIGS. 11A-F. N-terminal deletions of the all polypeptide of the invention shown as SEQ ID NO:187 include polypeptides comprising the amino acid sequence of residues: N-terminal deletions of the all polypeptide of the invention shown as SEQ ID NO:187 include polypeptides comprising the amino acid sequence of residues: D-2 to E-1189; L-3 to E-1189; P-4 to E-1189; R-5 to E-1189; G-6 to E-1189; L-7 to E-1189; V-8 to E-1189; V-9 to E-1189; A-10 to E-1189; W-11 to E-1189; A-12 to E-1189; L-13 to E-1189; S-14 to E-1189; L-15 to E-1189; W-16 to E-1189; P-17 to E-1189; G-18 to E-1189; F-19 to E-1189; T-20 to E-1189; D-21 to E-1189; T-22 to E-1189; F-23 to E-1189; N-24 to E-1189; M-25 to E-1189; D-26 to E-1189; T-27 to E-1189; R-28 to E-1189; K-29 to E-1189; P-30 to E-1189; R-31 to E-1189; V-32 to E-1189; 1-33 to E-1189; P-34 to E-1189; G-35 to E-1189; S-36 to E-1189; R-37 to E-1189; T-38 to E-1189; A-39 to E-1189; F-40 to E-1189; F-41 to E-1189; G-42 to E-1189; Y-43 to E-1189; T-44 to E-1189; V-45 to E-1189; Q-46 to E-1189; Q-47 to E-1189; H-48 to E-1189; D-49 to E-1189; I-50 to E-1189; S-51 to E-1189; G-52 to E-1189; N-53 to E-1189; K-54 to E-1189; W-55 to E-1189; L-56 to E-1189; V-57 to E-1189; V-58 to E-1189; G-59 to E-1189; A-60 to E-1189; P-61 to E-1189; L-62 to E-1189; E-63 to E-1189; T-64 to E-1189; N-65 to E-1189; G-66 to E-1189; Y-67 to E-1189; Q-68 to E-1189; K-69 to E-1189; T-70 to E-1189; G-71 to E-1189; D-72 to E-1189; V-73 to E-1189; Y-74 to E-1189; K-75 to E-1189; C-76 to E-1189; P-77 to E-1189; V-78 to E-1189; 1-79 to E-1189; H-80 to E-1189; G-81 to E-1189; N-82 to E-1189; C-83 to E-1189; T-84 to E-1189; K-85 to E-1189; L-86 to E-1189; N-87 to E-1189; L-88 to E-1189; G-89 to E-1189; R-90 to E-1189; V-91 to E-1189; T-92 to E-1189; L-93 to E-1189; S-94 to E-1189; N-95 to E-1189; V-96 to E-1189; S-97 to E-1189; E-98 to E-1189; R-99 to E-1189; K-100 to E-1189; D-101 to E-1189; N-102 to E-1189; M-103 to E-1189; R-104 to E-1189; L-105 to E-1189; G-106 to E-1189; L-107 to E-1189; S-108 to E-1189; L-109 to E-1189; A-110 to E-1189; T-111 to E-1189; N-112 to E-1189; P-113 to E-1189; K-114 to E-1189; D-115 to E-1189; N-116 to E-1189; S-117 to E-1189; F-118 to E-1189; L-119 to E-1189; A-120 to E-1189; C-121 to E-1189; S-122 to E-1189; P-123 to E-1189; L-124 to E-1189; W-125 to E-1189; S-126 to E-1189; H-127 to E-1189; E-128 to E-1189; C-129 to E-1189; G-130 to E-1189; S-131 to E-1189; S-132 to E-1189; Y-133 to E-1189; Y-134 to E-1189; T-135 to E-1189; T-136 to E-1189; G-137 to E-1189; M-138 to E-1189; C-139 to E-1189; S-140 to E-1189; R-141 to E-1189; V-142 to E-1189; N-143 to E-1189; S-144 to E-1189; N-145 to E-1189; F-146 to E-1189; R-147 to E-1189; F-148 to E-1189; S-149 to E-1189; K-150 to E-1189; T-151 to E-1189; V-152 to E-1189; A-153 to E-1189; P-154 to E-1189; A-155 to E-1189; L-156 to E-1189; Q-157 to E-1189; R-158 to E-1189; C-159 to E-1189; Q-160 to E-1189; T-161 to E-1189; Y-162 to E-1189; M-163 to E-1189; D-164 to E-1189; 1-165 to E-1189; V-166 to E-1189; 1-167 to E-1189; V-168 to E-1189; L-169 to E-1189; D-170 to E-1189; G-171 to E-1189; S-172 to E-1189; N-173 to E-1189; 5-174 to E-1189; 1-175 to E-1189; Y-176 to E-1189; P-177 to E-1189; W-178 to E-1189; V-179 to E-1189; E-180 to E-1189; V-181 to E-1189; Q-182 to E-1189; H-183 to E-1189; F-184 to E-1189; L-185 to E-1189; 1-186 to E-1189; N-187 to E-1189; 1-188 to E-1189; L-189 to E-1189; K-190 to E-1189; K-191 to E-1189; F-192 to E-1189; Y-193 to E-1189; I-194 to E-1189; G-195 to E-1189; P-196 to E-1189; G-197 to E-1189; Q-198 to E-1189; 1-199 to E-1189; Q-200 to E-1189; V-201 to E-1189; G-202 to E-1189; V-203 to E-1189; V-204 to E-1189; Q-205 to E-1189; Y-206 to E-1189; G-207 to E-1189; E-208 to E-1189; D-209 to E-1189; V-210 to E-1189; V-211 to E-1189; H-212 to E-1189; E-213 to E-1189; F-214 to E-1189; H-215 to E-1189; L-216 to E-1189; N-217 to E-1189; D-218 to E-1189; Y-219 to E-1189; R-220 to E-1189; S-221 to E-1189; V-222 to E-1189; K-223 to E-1189; D-224 to E-1189; V-225 to E-1189; V-226 to E-1189; E-227 to E-1189; A-228 to E-1189; A-229 to E-1189; S-230 to E-1189; H-231 to E-1189; I-232 to E-1189; E-233 to E-1189; Q-234 to E-1189; R-235 to E-1189; G-236 to E-1189; G-237 to E-1189; T-238 to E-1189; E-239 to E-1189; T-240 to E-1189; R-241 to E-1189; T-242 to E-1189; A-243 to E-1189; F-244 to E-1189; G-245 to E-1189; 1-246 to E-1189; E-247 to E-1189; F-248 to E-1189; A-249 to E-1189; R-250 to E-1189; S-251 to E-1189; E-252 to E-1189; A-253 to E-1189; F-254 to E-1189; Q-255 to E-1189; K-256 to E-1189; G-257 to E-1189; G-258 to E-1189; R-259 to E-1189; K-260 to E-1189; G-261 to E-1189; A-262 to E-1189; K-263 to E-1189; K-264 to E-1189; V-265 to E-1189; M-266 to E-1189; I-267 to E-1189; V-268 to E-1189; I-269 to E-1189; T-270 to E-1189; D-271 to E-1189; G-272 to E-1189; E-273 to E-1189; S-274 to E-1189; H-275 to E-1189; D-276 to E-1189; S-277 to E-1189; P-278 to E-1189; D-279 to E-1189; L-280 to E-1189; E-281 to E-1189; K-282 to E-1189; V-283 to E-1189; 1-284 to E-1189; Q-285 to E-1189; Q-286 to E-1189; S-287 to E-1189; E-288 to E-1189; R-289 to E-1189; D-290 to E-1189; N-291 to E-1189; V-292 to E-1189; T-293 to E-1189; R-294 to E-1189; Y-295 to E-1189; A-296 to E-1189; V-297 to E-1189; A-298 to E-1189; V-299 to E-1189; L-300 to E-1189; G-301 to E-1189; Y-302 to E-1189; Y-303 to E-1189; N-304 to E-1189; R-305 to E-1189; R-306 to E-1189; G-307 to E-1189; 1-308 to E-1189; N-309 to E-1189; P-310 to E-1189; E-311 to E-1189; T-312 to E-1189; F-313 to E-1189; L-314 to E-1189; N-315 to E-1189; E-316 to E-1189; 1-317 to E-1189; K-318 to E-1189; Y-319 to E-1189; I-320 to E-1189; A-321 to E-1189; S-322 to E-1189; D-323 to E-1189; P-324 to E-1189; D-325 to E-1189; D-326 to E-1189; K-327 to E-1189; H-328 to E-1189; F-329 to E-1189; F-330 to E-1189; N-331 to E-1189; V-332 to E-1189; T-333 to E-1189; D-334 to E-1189; E-335 to E-1189; A-336 to E-1189; A-337 to E-1189; L-338 to E-1189; K-339 to E-1189; D-340 to E-1189; 1-341 to E-1189; V-342 to E-1189; D-343 to E-1189; A-344 to E-1189; L-345 to E-1189; G-346 to E-1189; D-347 to E-1189; R-348 to E-1189; I-349 to E-1189; F-350 to E-1189; S-351 to E-1189; L-352 to E-1189; E-353 to E-1189; G-354 to E-1189; T-355 to E-1189; N-356 to E-1189; K-357 to E-1189; N-358 to E-1189; E-359 to E-1189; T-360 to E-1189; S-361 to E-1189; F-362 to E-1189; G-363 to E-1189; L-364 to E-1189; E-365 to E-1189; M-366 to E-1189; S-367 to E-1189; Q-368 to E-1189; T-369 to E-1189; G-370 to E-1189; F-371 to E-1189; S-372 to E-1189; S-373 to E-1189; H-374 to E-1189; V-375 to E-1189; V-376 to E-1189; E-377 to E-1189; D-378 to E-1189; G-379 to E-1189; V-380 to E-1189; L-381 to E-1189; L-382 to E-1189; G-383 to E-1189; A-384 to E-1189; V-385 to E-1189; G-386 to E-1189; A-387 to E-1189; Y-388 to E-1189; D-389 to E-1189; W-390 to E-1189; N-391 to E-1189; G-392 to E-1189; A-393 to E-1189; V-394 to E-1189; L-395 to E-1189; K-396 to E-1189; E-397 to E-1189; T-398 to E-1189; S-399 to E-1189; A-400 to E-1189; G-401 to E-1189; K-402 to E-1189; V-403 to E-1189; 1-404 to E-1189; P-405 to E-1189; L-406 to E-1189; R-407 to E-1189; E-408 to E-1189; S-409 to E-1189; Y-410 to E-1189; L-411 to E-1189; K-412 to E-1189; E-413 to E-1189; F-414 to E-1189; P-415 to E-1189; E-416 to E-1189; E-417 to E-1189; L-418 to E-1189; K-419 to E-1189; N-420 to E-1189; H-421 to E-1189; G-422 to E-1189; A-423 to E-1189; Y-424 to E-1189; L-425 to E-1189; G-426 to E-1189; Y-427 to E-1189; T-428 to E-1189; V-429 to E-1189; T-430 to E-1189; S-431 to E-1189; V-432 to E-1189; V-433 to E-1189; S-434 to E-1189; S-435 to E-1189; R-436 to E-1189; Q-437 to E-1189; G-438 to E-1189; R-439 to E-1189; V-440 to E-1189; Y-441 to E-1189; V-442 to E-1189; A-443 to E-1189; G-444 to E-1189; A-445 to E-1189; P-446 to E-1189; R-447 to E-1189; F-448 to E-1189; N-449 to E-1189; H-450 to E-1189; T-451 to E-1189; G-452 to E-1189; K-453 to E-1189; V-454 to E-1189; 1-455 to E-1189; L-456 to E-1189; F-457 to E-1189; T-458 to E-1189; M-459 to E-1189; H-460 to E-1189; N-461 to E-1189; N-462 to E-1189; R-463 to E-1189; S-464 to E-1189; L-465 to E-1189; T-466 to E-1189; I-467 to E-1189; H-468 to E-1189; Q-469 to E-1189; A-470 to E-1189; M-471 to E-1189; R-472 to E-1189; G-473 to E-1189; Q-474 to E-1189; Q-475 to E-1189; 1-476 to E-1189; G-477 to E-1189; S-478 to E-1189; Y-479 to E-1189; F-480 to E-1189; G-481 to E-1189; S-482 to E-1189; E-483 to E-1189; I-484 to E-1189; T-485 to E-1189; S-486 to E-1189; V-487 to E-1189; D-488 to E-1189; 1-489 to E-1189; D-490 to E-1189; G-491 to E-1189; D-492 to E-1189; G-493 to E-1189; V-494 to E-1189; T-495 to E-1189; D-496 to E-1189; V-497 to E-1189; L-498 to E-1189; L-499 to E-1189; V-500 to E-1189; G-501 to E-1189; A-502 to E-1189; P-503 to E-1189; M-504 to E-1189; Y-505 to E-1189; F-506 to E-1189; N-507 to E-1189; E-508 to E-1189; G-509 to E-1189; R-510 to E-1189; E-511 to E-1189; R-512 to E-1189; G-513 to E-1189; K-514 to E-1189; V-515 to E-1189; Y-516 to E-1189; V-517 to E-1189; Y-518 to E-1189; E-519 to E-1189; L-520 to E-1189; R-521 to E-1189; Q-522 to E-1189; N-523 to E-1189; R-524 to E-1189; F-525 to E-1189; V-526 to E-1189; Y-527 to E-1189; N-528 to E-1189; G-529 to E-1189; T-530 to E-1189; L-531 to E-1189; K-532 to E-1189; D-533 to E-1189; S-534 to E-1189; H-535 to E-1189; S-536 to E-1189; Y-537 to E-1189; Q-538 to E-1189; N-539 to E-1189; A-540 to E-1189; R-541 to E-1189; F-542 to E-1189; G-543 to E-1189; S-544 to E-1189; S-545 to E-1189; I-546 to E-1189; A-547 to E-1189; S-548 to E-1189; V-549 to E-1189; R-550 to E-1189; D-551 to E-1189; L-552 to E-1189; N-553 to E-1189; Q-554 to E-1189; D-555 to E-1189; S-556 to E-1189; Y-557 to E-1189; N-558 to E-1189; D-559 to E-1189; V-560 to E-1189; V-561 to E-1189; V-562 to E-1189; G-563 to E-1189; A-564 to E-1189; P-565 to E-1189; L-566 to E-1189; E-567 to E-1189; D-568 to E-1189; N-569 to E-1189; H-570 to E-1189; A-571 to E-1189; G-572 to E-1189; A-573 to E-1189; 1-574 to E-1189; Y-575 to E-1189; 1-576 to E-1189; F-577 to E-1189; H-578 to E-1189; G-579 to E-1189; F-580 to E-1189; R-581 to E-1189; G-582 to E-1189; S-583 to E-1189; 1-584 to E-1189; L-585 to E-1189; K-586 to E-1189; T-587 to E-1189; P-588 to E-1189; K-589 to E-1189; Q-590 to E-1189; R-591 to E-1189; 1-592 to E-1189; T-593 to E-1189; A-594 to E-1189; S-595 to E-1189; E-596 to E-1189; L-597 to E-1189; A-598 to E-1189; T-599 to E-1189; G-600 to E-1189; L-601 to E-1189; Q-602 to E-1189; Y-603 to E-1189; F-604 to E-1189; G-605 to E-1189; C-606 to E-1189; S-607 to E-1189; 1-608 to E-1189; H-609 to E-1189; G-610 to E-1189; Q-611 to E-1189; L-612 to E-1189; D-613 to E-1189; L-614 to E-1189; N-615 to E-1189; E-616 to E-1189; D-617 to E-1189; G-618 to E-1189; L-619 to E-1189; 1-620 to E-1189; D-621 to E-1189; L-622 to E-1189; A-623 to E-1189; V-624 to E-1189; G-625 to E-1189; A-626 to E-1189; L-627 to E-1189; G-628 to E-1189; N-629 to E-1189; A-630 to E-1189; V-631 to E-1189; I-632 to E-1189; L-633 to E-1189; W-634 to E-1189; S-635 to E-1189; R-636 to E-1189; P-637 to E-1189; V-638 to E-1189; V-639 to E-1189; Q-640 to E-1189; 1-641 to E-1189; N-642 to E-1189; A-643 to E-1189; S-644 to E-1189; L-645 to E-1189; H-646 to E-1189; F-647 to E-1189; E-648 to E-1189; P-649 to E-1189; S-650 to E-1189; K-651 to E-1189; 1-652 to E-1189; N-653 to E-1189; 1-654 to E-1189; F-655 to E-1189; H-656 to E-1189; R-657 to E-1189; D-658 to E-1189; C-659 to E-1189; K-660 to E-1189; R-661 to E-1189; S-662 to E-1189; G-663 to E-1189; R-664 to E-1189; D-665 to E-1189; A-666 to E-1189; T-667 to E-1189; C-668 to E-1189; L-669 to E-1189; A-670 to E-1189; A-671 to E-1189; F-672 to E-1189; L-673 to E-1189; C-674 to E-1189; F-675 to E-1189; T-676 to E-1189; P-677 to E-1189; 1-678 to E-1189; F-679 to E-1189; L-680 to E-1189; A-681 to E-1189; P-682 to E-1189; H-683 to E-1189; F-684 to E-1189; Q-685 to E-1189; T-686 to E-1189; T-687 to E-1189; T-688 to E-1189; V-689 to E-1189; G-690 to E-1189; 1-691 to E-1189; R-692 to E-1189; Y-693 to E-1189; N-694 to E-1189; A-695 to E-1189; T-696 to E-1189; M-697 to E-1189; D-698 to E-1189; E-699 to E-1189; R-700 to E-1189; R-701 to E-1189; Y-702 to E-1189; T-703 to E-1189; P-704 to E-1189; R-705 to E-1189; A-706 to E-1189; H-707 to E-1189; L-708 to E-1189; D-709 to E-1189; E-710 to E-1189; G-711 to E-1189; G-712 to E-1189; D-713 to E-1189; R-714 to E-1189; F-715 to E-1189; T-716 to E-1189; N-717 to E-1189; R-718 to E-1189; A-719 to E-1189; V-720 to E-1189; L-721 to E-1189; L-722 to E-1189; S-723 to E-1189; S-724 to E-1189; G-725 to E-1189; Q-726 to E-1189; E-727 to E-1189; L-728 to E-1189; C-729 to E-1189; E-730 to E-1189; R-731 to E-1189; 1-732 to E-1189; N-733 to E-1189; F-734 to E-1189; H-735 to E-1189; V-736 to E-1189; L-737 to E-1189; D-738 to E-1189; T-739 to E-1189; A-740 to E-1189; D-741 to E-1189; Y-742 to E-1189; V-743 to E-1189; K-744 to E-1189; P-745 to E-1189; V-746 to E-1189; T-747 to E-1189; F-748 to E-1189; S-749 to E-1189; V-750 to E-1189; E-751 to E-1189; Y-752 to E-1189; S-753 to E-1189; L-754 to E-1189; E-755 to E-1189; D-756 to E-1189; P-757 to E-1189; D-758 to E-1189; H-759 to E-1189; G-760 to E-1189; P-761 to E-1189; M-762 to E-1189; L-763 to E-1189; D-764 to E-1189; D-765 to E-1189; G-766 to E-1189; W-767 to E-1189; P-768 to E-1189; T-769 to E-1189; T-770 to E-1189; L-771 to E-1189; R-772 to E-1189; V-773 to E-1189; S-774 to E-1189; V-775 to E-1189; P-776 to E-1189; F-777 to E-1189; W-778 to E-1189; N-779 to E-1189; G-780 to E-1189; C-781 to E-1189; N-782 to E-1189; E-783 to E-1189; D-784 to E-1189; E-785 to E-1189; H-786 to E-1189; C-787 to E-1189; V-788 to E-1189; P-789 to E-1189; D-790 to E-1189; L-791 to E-1189; V-792 to E-1189; L-793 to E-1189; D-794 to E-1189; A-795 to E-1189; R-796 to E-1189; S-797 to E-1189; D-798 to E-1189; L-799 to E-1189; P-800 to E-1189; T-801 to E-1189; A-802 to E-1189; M-803 to E-1189; E-804 to E-1189; Y-805 to E-1189; C-806 to E-1189; Q-807 to E-1189; R-808 to E-1189; V-809 to E-1189; L-810 to E-1189; R-811 to E-1189; K-812 to E-1189; P-813 to E-1189; A-814 to E-1189; Q-815 to E-1189; D-816 to E-1189; C-817 to E-1189; S-818 to E-1189; A-819 to E-1189; Y-820 to E-1189; T-821 to E-1189; L-822 to E-1189; S-823 to E-1189; F-824 to E-1189; D-825 to E-1189; T-826 to E-1189; T-827 to E-1189; V-828 to E-1189; F-829 to E-1189; I-830 to E-1189; 1-831 to E-1189; E-832 to E-1189; S-833 to E-1189; T-834 to E-1189; R-835 to E-1189; Q-836 to E-1189; R-837 to E-1189; V-838 to E-1189; A-839 to E-1189; V-840 to E-1189; E-841 to E-1189; A-842 to E-1189; T-843 to E-1189; L-844 to E-1189; E-845 to E-1189; N-846 to E-1189; R-847 to E-1189; G-848 to E-1189; E-849 to E-1189; N-850 to E-1189; A-851 to E-1189; Y-852 to E-1189; S-853 to E-1189; T-854 to E-1189; V-855 to E-1189; L-856 to E-1189; N-857 to E-1189; 1-858 to E-1189; S-859 to E-1189; Q-860 to E-1189; S-861 to E-1189; A-862 to E-1189; N-863 to E-1189; L-864 to E-1189; Q-865 to E-1189; F-866 to E-1189; A-867 to E-1189; S-868 to E-1189; L-869 to E-1189; 1-870 to E-1189; Q-871 to E-1189; K-872 to E-1189; E-873 to E-1189; D-874 to E-1189; S-875 to E-1189; D-876 to E-1189; G-877 to E-1189; S-878 to E-1189; 1-879 to E-1189; E-880 to E-1189; C-881 to E-1189; V-882 to E-1189; N-883 to E-1189; E-884 to E-1189; E-885 to E-1189; R-886 to E-1189; R-887 to E-1189; L-888 to E-1189; Q-889 to E-1189; K-890 to E-1189; Q-891 to E-1189; V-892 to E-1189; C-893 to E-1189; N-894 to E-1189; V-895 to E-1189; S-896 to E-1189; Y-897 to E-1189; P-898 to E-1189; F-899 to E-1189; F-900 to E-1189; R-901 to E-1189; A-902 to E-1189; K-903 to E-1189; A-904 to E-1189; K-905 to E-1189; V-906 to E-1189; A-907 to E-1189; F-908 to E-1189; R-909 to E-1189; L-910 to E-1189; D-911 to E-1189; F-912 to E-1189; E-913 to E-1189; F-914 to E-1189; S-915 to E-1189; K-916 to E-1189; S-917 to E-1189; 1-918 to E-1189; F-919 to E-1189; L-920 to E-1189; H-921 to E-1189; H-922 to E-1189; L-923 to E-1189; E-924 to E-1189; 1-925 to E-1189; E-926 to E-1189; L-927 to E-1189; A-928 to E-1189; A-929 to E-1189; G-930 to E-1189; S-931 to E-1189; D-932 to E-1189; S-933 to E-1189; N-934 to E-1189; E-935 to E-1189; R-936 to E-1189; D-937 to E-1189; S-938 to E-1189; T-939 to E-1189; K-940 to E-1189; E-941 to E-1189; D-942 to E-1189; N-943 to E-1189; V-944 to E-1189; A-945 to E-1189; P-946 to E-1189; L-947 to E-1189; R-948 to E-1189; F-949 to E-1189; H-950 to E-1189; L-951 to E-1189; K-952 to E-1189; Y-953 to E-1189; E-954 to E-1189; A-955 to E-1189; D-956 to E-1189; V-957 to E-1189; L-958 to E-1189; F-959 to E-1189; T-960 to E-1189; R-961 to E-1189; S-962 to E-1189; S-963 to E-1189; S-964 to E-1189; L-965 to E-1189; S-966 to E-1189; H-967 to E-1189; Y-968 to E-1189; E-969 to E-1189; V-970 to E-1189; K-971 to E-1189; L-972 to E-1189; N-973 to E-1189; S-974 to E-1189; S-975 to E-1189; L-976 to E-1189; E-977 to E-1189; R-978 to E-1189; Y-979 to E-1189; D-980 to E-1189; G-981 to E-1189; 1-982 to E-1189; G-983 to E-1189; P-984 to E-1189; P-985 to E-1189; F-986 to E-1189; S-987 to E-1189; C-988 to E-1189; 1-989 to E-1189; F-990 to E-1189; R-991 to E-1189; 1-992 to E-1189; Q-993 to E-1189; N-994 to E-1189; L-995 to E-1189; G-996 to E-1189; L-997 to E-1189; F-998 to E-1189; P-999 to E-1189; I-1000 to E-1189; H-1001 to E-1189; G-1002 to E-1189; 1-1003 to E-1189; M-1004 to E-1189; M-1005 to E-1189; K-1006 to E-1189; 1-1007 to E-1189; T-1008 to E-1189; I-1009 to E-1189; P-1010 to E-1189; I-1011 to E-1189; A-1012 to E-1189; T-1013 to E-1189; R-1014 to E-1189; S-1015 to E-1189; G-1016 to E-1189; N-1017 to E-1189; R-1018 to E-1189; L-1019 to E-1189; L-1020 to E-1189; K-1021 to E-1189; L-1022 to E-1189; R-1023 to E-1189; D-1024 to E-1189; F-1025 to E-1189; L-1026 to E-1189; T-1027 to E-1189; D-1028 to E-1189; E-1029 to E-1189; V-1030 to E-1189; A-1031 to E-1189; N-1032 to E-1189; T-1033 to E-1189; S-1034 to E-1189; C-1035 to E-1189; N-1036 to E-1189; 1-1037 to E-1189; W-1038 to E-1189; G-1039 to E-1189; N-1040 to E-1189; S-1041 to E-1189; T-1042 to E-1189; E-1043 to E-1189; Y-1044 to E-1189; R-1045 to E-1189; P-1046 to E-1189; T-1047 to E-1189; P-1048 to E-1189; V-1049 to E-1189; E-1050 to E-1189; E-1051 to E-1189; D-1052 to E-1189; L-1053 to E-1189; R-1054 to E-1189; R-1055 to E-1189; A-1056 to E-1189; P-1057 to E-1189; Q-1058 to E-1189; L-1059 to E-1189; N-1060 to E-1189; H-1061 to E-1189; S-1062 to E-1189; N-1063 to E-1189; S-1064 to E-1189; D-1065 to E-1189; V-1066 to E-1189; V-1067 to E-1189; S-1068 to E-1189; 1-1069 to E-1189; N-1070 to E-1189; C-1071 to E-1189; N-1072 to E-1189; 1-1073 to E-1189; R-1074 to E-1189; L-1075 to E-1189; V-1076 to E-1189; P-1077 to E-1189; N-1078 to E-1189; Q-1079 to E-1189; E-1080 to E-1189; 1-1081 to E-1189; N-1082 to E-1189; F-1083 to E-1189; H-1084 to E-1189; L-1085 to E-1189; L-1086 to E-1189; G-1087 to E-1189; N-1088 to E-1189; L-1089 to E-1189; W-1090 to E-1189; L-1091 to E-1189; R-1092 to E-1189; S-1093 to E-1189; L-1094 to E-1189; K-1095 to E-1189; A-1096 to E-1189; L-1097 to E-1189; K-1098 to E-1189; Y-1099 to E-1189; K-1100 to E-1189; S-1101 to E-1189; M-1102 to E-1189; K-1103 to E-1189; 1-1104 to E-1189; M-1105 to E-1189; V-1106 to E-1189; N-1107 to E-1189; A-1108 to E-1189; A-1109 to E-1189; L-1110 to E-1189; Q-1111 to E-1189; R-1112 to E-1189; Q-1113 to E-1189; F-1114 to E-1189; H-1115 to E-1189; 5-1116 to E-1189; P-1117 to E-1189; F-1118 to E-1189; 1-1119 to E-1189; F-1120 to E-1189; R-1121 to E-1189; E-1122 to E-1189; E-1123 to E-1189; D-1124 to E-1189; P-1125 to E-1189; 5-1126 to E-1189; R-1127 to E-1189; Q-1128 to E-1189; 1-1129 to E-1189; V-1130 to E-1189; F-1131 to E-1189; E-1132 to E-1189; 1-1133 to E-1189; 5-1134 to E-1189; K-1135 to E-1189; Q-1136 to E-1189; E-1137 to E-1189; D-1138 to E-1189; W-1139 to E-1189; Q-1140 to E-1189; V-1141 to E-1189; P-1142 to E-1189; 1-1143 to E-1189; W-1144 to E-1189; I-1145 to E-1189; 1-1146 to E-1189; V-1147 to E-1189; G-1148 to E-1189; 5-1149 to E-1189; T-1150 to E-1189; L-1151 to E-1189; G-1152 to E-1189; G-1153 to E-1189; L-1154 to E-1189; L-1155 to E-1189; L-1156 to E-1189; L-1157 to E-1189; A-1158 to E-1189; L-1159 to E-1189; L-1160 to E-1189; V-1161 to E-1189; L-1162 to E-1189; A-1163 to E-1189; L-1164 to E-1189; W-1165 to E-1189; K-1166 to E-1189; L-1167 to E-1189; G-1168 to E-1189; F-1169 to E-1189; F-1170 to E-1189; R-1171 to E-1189; 5-1172 to E-1189; A-1173 to E-1189; R-1174 to E-1189; R-1175 to E-1189; R-1176 to E-1189; R-1177 to E-1189; E-1178 to E-1189; P-1179 to E-1189; G-1180 to E-1189; L-1181 to E-1189; D-1182 to E-1189; P-1183 to E-1189; T-1184 to E-1189; of SEQ ID NO:187. Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (e.g., fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind these fragments and variants of the invention are also encompassed by the invention. Polynucleotides encoding these fragments and variants are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities (e.g., ability to illicit mitogenic activity, induce differentiation of normal or malignant cells, ability to multimerize, etc.) may still be retained. For example the ability of the shortened all mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an all mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six all amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the a11 polypeptide shown in FIGS. 11A-F, up to the glycine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising the amino acid sequence of residues 1-m1 of FIGS. 11A-F, where m1 is an integer from 6 to 1189 corresponding to the position of the amino acid residue in FIGS. 11A-F. Moreover, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of C-terminal deletions of the a11 polypeptide of the invention shown as SEQ ID NO:187 include polypeptides comprising the amino acid sequence of residues: M-1 to L-1188; M-1 to V-1187; M-1 to K-1186; M-1 to P-1185; M-1 to T-1184; M-1 to P-1183; M-1 to D-1182; M-1 to L-1181; M-1 to G-1180; M-1 to P-1179; M-1 to E-1178; M-1 to R-1177; M-1 to R-1176; M-1 to R-1175; M-1 to R-1174; M-1 to A-1173; M-1 to S-1172; M-1 to R-1171; M-1 to F-1170; M-1 to F-1169; M-1 to G-1168; M-1 to L-1167; M-1 to K-1166; M-1 to W-1165; M-1 to L-1164; M-1 to A-1163; M-1 to L-1162; M-1 to V-1161; M-1 to L-1160; M-1 to L-1159; M-1 to A-1158; M-1 to L-1157; M-1 to L-1156; M-1 to L-1155; M-1 to L-1154; M-1 to G-1153; M-1 to G-1152; M-1 to L-1151; M-1 to T-1150; M-1 to S-1149; M-1 to G-1148; M-1 to V-1147; M-1 to I-1146; M-1 to I-1145; M-1 to W-1144; M-1 to I-1143; M-1 to P-1142; M-1 to V-1141; M-1 to Q-1140; M-1 to W-1139; M-1 to D-1138; M-1 to E-1137; M-1 to Q-1136; M-1 to K-1135; M-1 to S-1134; M-1 to I-1133; M-1 to E-1132; M-1 to F-1131; M-1 to V-1130; M-1 to I-1129; M-1 to Q-1128; M-1 to R-1127; M-1 to S-1126; M-1 to P-1125; M-1 to D-1124; M-1 to E-1123; M-1 to E-1122; M-1 to R-1121; M-1 to F-1120; M-1 to I-1119; M-1 to F-1118; M-1 to P-1117; M-1 to S-1116; M-1 to H-1115; M-1 to F-1114; M-1 to Q-1113; M-1 to R-1112; M-1 to Q-1111; M-1 to L-1110; M-1 to A-1109; M-1 to A-1108; M-1 to N-1107; M-1 to V-1106; M-1 to M-1105; M-1 to I-1104; M-1 to K-1103; M-1 to M-1102; M-1 to S-1101; M-1 to K-1100; M-1 to Y-1099; M-1 to K-1098; M-1 to L-1097; M-1 to A-1096; M-1 to K-1095; M-1 to L-1094; M-1 to S-1093; M-1 to R-1092; M-1 to L-1091; M-1 to W-1090; M-1 to L-1089; M-1 to N-1088; M-1 to G-1087; M-1 to L-1086; M-1 to L-1085; M-1 to H-1084; M-1 to F-1083; M-1 to N-1082; M-1 to I-1081; M-1 to E-1080; M-1 to Q-1079; M-1 to N-1078; M-1 to P-1077; M-1 to V-1076; M-1 to L-1075; M-1 to R-1074; M-1 to I-1073; M-1 to N-1072; M-1 to C-1071; M-1 to N-1070; M-1 to I-1069; M-1 to S-1068; M-1 to V-1067; M-1 to V-1066; M-1 to D-1065; M-1 to S-1064; M-1 to N-1063; M-1 to S-1062; M-1 to H-1061; M-1 to N-1060; M-1 to L-1059; M-1 to Q-1058; M-1 to P-1057; M-1 to A-1056; M-1 to R-1055; M-1 to R-1054; M-1 to L-1053; M-1 to D-1052; M-1 to E-1051; M-1 to E-1050; M-1 to V-1049; M-1 to P-1048; M-1 to T-1047; M-1 to P-1046; M-1 to R-1045; M-1 to Y-1044; M-1 to E-1043; M-1 to T-1042; M-1 to S-1041; M-1 to N-1040; M-1 to G-1039; M-1 to W-1038; M-1 to I-1037; M-1 to N-1036; M-1 to C-1035; M-1 to S-1034; M-1 to T-1033; M-1 to N-1032; M-1 to A-1031; M-1 to V-1030; M-1 to E-1029; M-1 to D-1028; M-1 to T-1027; M-1 to L-1026; M-1 to F-1025; M-1 to D-1024; M-1 to R-1023; M-1 to L-1022; M-1 to K-1021; M-1 to L-1020; M-1 to L-1019; M-1 to R-1018; M-1 to N-1017; M-1 to G-1016; M-1 to S-1015; M-1 to R-1014; M-1 to T-1013; M-1 to A-1012; M-1 to I-1011; M-1 to P-1010; M-1 to I-1009; M-1 to T-1008; M-1 to I-1007; M-1 to K-1006; M-1 to M-1005; M-1 to M-1004; M-1 to I-1003; M-1 to G-1002; M-1 to H-1001; M-1 to I-1000; M-1 to P-999; M-1 to F-998; M-1 to L-997; M-1 to G-996; M-1 to L-995; M-1 to N-994; M-1 to Q-993; M-1 to I-992; M-1 to R-991; M-1 to F-990; M-1 to I-989; M-1 to C-988; M-1 to S-987; M-1 to F-986; M-1 to P-985; M-1 to P-984; M-1 to G-983; M-1 to I-982; M-1 to G-981; M-1 to D-980; M-1 to Y-979; M-1 to R-978; M-1 to E-977; M-1 to L-976; M-1 to S-975; M-1 to S-974; M-1 to N-973; M-1 to L-972; M-1 to K-971; M-1 to V-970; M-1 to E-969; M-1 to Y-968; M-1 to H-967; M-1 to S-966; M-1 to L-965; M-1 to S-964; M-1 to S-963; M-1 to S-962; M-1 to R-961; M-1 to T-960; M-1 to F-959; M-1 to L-958; M-1 to V-957; M-1 to D-956; M-1 to A-955; M-1 to E-954; M-1 to Y-953; M-1 to K-952; M-1 to L-951; M-1 to H-950; M-1 to F-949; M-1 to R-948; M-1 to L-947; M-1 to P-946; M-1 to A-945; M-1 to V-944; M-1 to N-943; M-1 to D-942; M-1 to E-941; M-1 to K-940; M-1 to T-939; M-1 to S-938; M-1 to D-937; M-1 to R-936; M-1 to E-935; M-1 to N-934; M-1 to S-933; M-1 to D-932; M-1 to S-931; M-1 to G-930; M-1 to A-929; M-1 to A-928; M-1 to L-927; M-1 to E-926; M-1 to I-925; M-1 to E-924; M-1 to L-923; M-1 to H-922; M-1 to H-921; M-1 to L-920; M-1 to F-919; M-1 to I-918; M-1 to S-917; M-1 to K-916; M-1 to S-915; M-1 to F-914; M-1 to E-913; M-1 to F-912; M-1 to D-911; M-1 to L-910; M-1 to R-909; M-1 to F-908; M-1 to A-907; M-1 to V-906; M-1 to K-905; M-1 to A-904; M-1 to K-903; M-1 to A-902; M-1 to R-901; M-1 to F-900; M-1 to F-899; M-1 to P-898; M-1 to Y-897; M-1 to S-896; M-1 to V-895; M-1 to N-894; M-1 to C-893; M-1 to V-892; M-1 to Q-891; M-1 to K-890; M-1 to Q-889; M-1 to L-888; M-1 to R-887; M-1 to R-886; M-1 to E-885; M-1 to E-884; M-1 to N-883; M-1 to V-882; M-1 to C-881; M-1 to E-880; M-1 to I-879; M-1 to S-878; M-1 to G-877; M-1 to D-876; M-1 to S-875; M-1 to D-874; M-1 to E-873; M-1 to K-872; M-1 to Q-871; M-1 to I-870; M-1 to L-869; M-1 to S-868; M-1 to A-867; M-1 to F-866; M-1 to Q-865; M-1 to L-864; M-1 to N-863; M-1 to A-862; M-1 to S-861; M-1 to Q-860; M-1 to S-859; M-1 to I-858; M-1 to N-857; M-1 to L-856; M-1 to V-855; M-1 to T-854; M-1 to S-853; M-1 to Y-852; M-1 to A-851; M-1 to N-850; M-1 to E-849; M-1 to G-848; M-1 to R-847; M-1 to N-846; M-1 to E-845; M-1 to L-844; M-1 to T-843; M-1 to A-842; M-1 to E-841; M-1 to V-840; M-1 to A-839; M-1 to V-838; M-1 to R-837; M-1 to Q-836; M-1 to R-835; M-1 to T-834; M-1 to S-833; M-1 to E-832; M-1 to I-831; M-1 to I-830; M-1 to F-829; M-1 to V-828; M-1 to T-827; M-1 to T-826; M-1 to D-825; M-1 to F-824; M-1 to S-823; M-1 to L-822; M-1 to T-821; M-1 to Y-820; M-1 to A-819; M-1 to S-818; M-1 to C-817; M-1 to D-816; M-1 to Q-815; M-1 to A-814; M-1 to P-813; M-1 to K-812; M-1 to R-811; M-1 to L-810; M-1 to V-809; M-1 to R-808; M-1 to Q-807; M-1 to C-806; M-1 to Y-805; M-1 to E-804; M-1 to M-803; M-1 to A-802; M-1 to T-801; M-1 to P-800; M-1 to L-799; M-1 to D-798; M-1 to S-797; M-1 to R-796; M-1 to A-795; M-1 to D-794; M-1 to L-793; M-1 to V-792; M-1 to L-791; M-1 to D-790; M-1 to P-789; M-1 to V-788; M-1 to C-787; M-1 to H-786; M-1 to E-785; M-1 to D-784; M-1 to E-783; M-1 to N-782; M-1 to C-781; M-1 to G-780; M-1 to N-779; M-1 to W-778; M-1 to F-777; M-1 to P-776; M-1 to V-775; M-1 to S-774; M-1 to V-773; M-1 to R-772; M-1 to L-771; M-1 to T-770; M-1 to T-769; M-1 to P-768; M-1 to W-767; M-1 to G-766; M-1 to D-765; M-1 to D-764; M-1 to L-763; M-1 to M-762; M-1 to P-761; M-1 to G-760; M-1 to H-759; M-1 to D-758; M-1 to P-757; M-1 to D-756; M-1 to E-755; M-1 to L-754; M-1 to S-753; M-1 to Y-752; M-1 to E-751; M-1 to V-750; M-1 to S-749; M-1 to F-748; M-1 to T-747; M-1 to V-746; M-1 to P-745; M-1 to K-744; M-1 to V-743; M-1 to Y-742; M-1 to D-741; M-1 to A-740; M-1 to T-739; M-1 to D-738; M-1 to L-737; M-1 to V-736; M-1 to H-735; M-1 to F-734; M-1 to N-733; M-1 to I-732; M-1 to R-731; M-1 to E-730; M-1 to C-729; M-1 to L-728; M-1 to E-727; M-1 to Q-726; M-1 to G-725; M-1 to S-724; M-1 to S-723; M-1 to L-722; M-1 to L-721; M-1 to V-720; M-1 to A-719; M-1 to R-718; M-1 to N-717; M-1 to T-716; M-1 to F-715; M-1 to R-714; M-1 to D-713; M-1 to G-712; M-1 to G-711; M-1 to E-710; M-1 to D-709; M-1 to L-708; M-1 to H-707; M-1 to A-706; M-1 to R-705; M-1 to P-704; M-1 to T-703; M-1 to Y-702; M-1 to R-701; M-1 to R-700; M-1 to E-699; M-1 to D-698; M-1 to M-697; M-1 to T-696; M-1 to A-695; M-1 to N-694; M-1 to Y-693; M-1 to R-692; M-1 to I-691; M-1 to G-690; M-1 to V-689; M-1 to T-688; M-1 to T-687; M-1 to T-686; M-1 to Q-685; M-1 to F-684; M-1 to H-683; M-1 to P-682; M-1 to A-681; M-1 to L-680; M-1 to F-679; M-1 to I-678; M-1 to P-677; M-1 to T-676; M-1 to F-675; M-1 to C-674; M-1 to L-673; M-1 to F-672; M-1 to A-671; M-1 to A-670; M-1 to L-669; M-1 to C-668; M-1 to T-667; M-1 to A-666; M-1 to D-665; M-1 to R-664; M-1 to G-663; M-1 to S-662; M-1 to R-661; M-1 to K-660; M-1 to C-659; M-1 to D-658; M-1 to R-657; M-1 to H-656; M-1 to F-655; M-1 to I-654; M-1 to N-653; M-1 to I-652; M-1 to K-651; M-1 to S-650; M-1 to P-649; M-1 to E-648; M-1 to F-647; M-1 to H-646; M-1 to L-645; M-1 to S-644; M-1 to A-643; M-1 to N-642; M-1 to I-641; M-1 to Q-640; M-1 to V-639; M-1 to V-638; M-1 to P-637; M-1 to R-636; M-1 to S-635; M-1 to W-634; M-1 to L-633; M-1 to I-632; M-1 to V-631; M-1 to A-630; M-1 to N-629; M-1 to G-628; M-1 to L-627; M-1 to A-626; M-1 to G-625; M-1 to V-624; M-1 to A-623; M-1 to L-622; M-1 to D-621; M-1 to I-620; M-1 to L-619; M-1 to G-618; M-1 to D-617; M-1 to E-616; M-1 to N-615; M-1 to L-614; M-1 to D-613; M-1 to L-612; M-1 to Q-611; M-1 to G-610; M-1 to H-609; M-1 to I-608; M-1 to S-607; M-1 to C-606; M-1 to G-605; M-1 to F-604; M-1 to Y-603; M-1 to Q-602; M-1 to L-601; M-1 to G-600; M-1 to T-599; M-1 to A-598; M-1 to L-597; M-1 to E-596; M-1 to S-595; M-1 to A-594; M-1 to T-593; M-1 to I-592; M-1 to R-591; M-1 to Q-590; M-1 to K-589; M-1 to P-588; M-1 to T-587; M-1 to K-586; M-1 to L-585; M-1 to I-584; M-1 to S-583; M-1 to G-582; M-1 to R-581; M-1 to F-580; M-1 to G-579; M-1 to H-578; M-1 to F-577; M-1 to I-576; M-1 to Y-575; M-1 to I-574; M-1 to A-573; M-1 to G-572; M-1 to A-571; M-1 to H-570; M-1 to N-569; M-1 to D-568; M-1 to E-567; M-1 to L-566; M-1 to P-565; M-1 to A-564; M-1 to G-563; M-1 to V-562; M-1 to V-561; M-1 to V-560; M-1 to D-559; M-1 to N-558; M-1 to Y-557; M-1 to S-556; M-1 to D-555; M-1 to Q-554; M-1 to N-553; M-1 to L-552; M-1 to D-551; M-1 to R-550; M-1 to V-549; M-1 to S-548; M-1 to A-547; M-1 to I-546; M-1 to S-545; M-1 to S-544; M-1 to G-543; M-1 to F-542; M-1 to R-541; M-1 to A-540; M-1 to N-539; M-1 to Q-538; M-1 to Y-537; M-1 to S-536; M-1 to H-535; M-lto S-534; M-1 to D-533; M-1 to K-532; M-1 to L-531; M-1 to T-530; M-1 to G-529; M-1 to N-528; M-1 to Y-527; M-1 to V-526; M-1 to F-525; M-1 to R-524; M-1 to N-523; M-1 to Q-522; M-1 to R-521; M-1 to L-520; M-1 to E-519; M-1 to Y-518; M-1 to V-517; M-1 to Y-516; M-1 to V-515; M-1 to K-514; M-1 to G-513; M-1 to R-512; M-1 to E-511; M-1 to R-510; M-1 to G-509; M-1 to E-508; M-1 to N-507; M-1 to F-506; M-1 to Y-505; M-1 to M-504; M-1 to P-503; M-1 to A-502; M-1 to G-501; M-1 to V-500; M-1 to L-499; M-1 to L-498; M-1 to V-497; M-1 to D-496; M-1 to T-495; M-1 to V-494; M-1 to G-493; M-1 to D-492; M-1 to G-491; M-1 to D-490; M-1 to I-489; M-1 to D-488; M-1 to V-487; M-1 to S-486; M-1 to T-485; M-1 to I-484; M-1 to E-483; M-1 to S-482; M-1 to G-481; M-1 to F-480; M-1 to Y-479; M-1 to S-478; M-1 to G-477; M-1 to I-476; M-1 to Q-475; M-1 to Q-474; M-1 to G-473; M-1 to R-472; M-1 to M-471; M-1 to A-470; M-1 to Q-469; M-1 to H-468; M-1 to I-467; M-1 to T-466; M-1 to L-465; M-1 to S-464; M-1 to R-463; M-1 to N-462; M-1 to N-461; M-1 to H-460; M-1 to M-459; M-1 to T-458; M-1 to F-457; M-1 to L-456; M-1 to I-455; M-1 to V-454; M-1 to K-453; M-1 to G-452; M-1 to T-451; M-1 to H-450; M-1 to N-449; M-1 to F-448; M-1 to R-447; M-1 to P-446; M-1 to A-445; M-1 to G-444; M-1 to A-443; M-1 to V-442; M-1 to Y-441; M-1 to V-440; M-1 to R-439; M-1 to G-438; M-1 to Q-437; M-1 to R-436; M-1 to S-435; M-1 to S-434; M-1 to V-433; M-1 to V-432; M-1 to S-431; M-1 to T-430; M-1 to V-429; M-1 to T-428; M-1 to Y-427; M-1 to G-426; M-1 to L-425; M-1 to Y-424; M-1 to A-423; M-1 to G-422; M-1 to H-421; M-1 to N-420; M-1 to K-419; M-1 to L-418; M-1 to E-417; M-1 to E-416; M-1 to P-415; M-1 to F-414; M-1 to E-413; M-1 to K-412; M-1 to L-411; M-1 to Y-410; M-1 to S-409; M-1 to E-408; M-1 to R-407; M-1 to L-406; M-1 to P-405; M-1 to I-404; M-1 to V-403; M-1 to K-402; M-1 to G-401; M-1 to A-400; M-1 to S-399; M-1 to T-398; M-1 to E-397; M-1 to K-396; M-1 to L-395; M-1 to V-394; M-1 to A-393; M-1 to G-392; M-1 to N-391; M-1 to W-390; M-1 to D-389; M-1 to Y-388; M-1 to A-387; M-1 to G-386; M-1 to V-385; M-1 to A-384; M-1 to G-383; M-1 to L-382; M-1 to L-381; M-1 to V-380; M-1 to G-379; M-1 to D-378; M-1 to E-377; M-1 to V-376; M-1 to V-375; M-1 to H-374; M-1 to S-373; M-1 to S-372; M-1 to F-371; M-1 to G-370; M-1 to T-369; M-1 to Q-368; M-1 to S-367; M-1 to M-366; M-1 to E-365; M-1 to L-364; M-1 to G-363; M-1 to F-362; M-1 to S-361; M-1 to T-360; M-1 to E-359; M-1 to N-358; M-1 to K-357; M-1 to N-356; M-1 toT-355; M-1 to G-354; M-1 to E-353; M-1 to L-352; M-1 to S-351; M-1 to F-350; M-1 to I-349; M-1 to R-348; M-1 to D-347; M-1 to G-346; M-1 to L-345; M-1 to A-344; M-1 to D-343; M-1 to V-342; M-1 to I-341; M-1 to D-340; M-1 to K-339; M-1 to L-338; M-1 to A-337; M-1 to A-336; M-1 to E-335; M-1 to D-334; M-1 to T-333; M-1 to V-332; M-1 to N-331; M-1 to F-330; M-1 to F-329; M-1 to H-328; M-1 to K-327; M-1 to D-326; M-1 to D-325; M-1 to P-324; M-1 to D-323; M-1 to S-322; M-1 to A-321; M-1 to I-320; M-1 to Y-319; M-1 to K-318; M-1 to I-317; M-1 to E-316; M-1 to N-315; M-1 to L-314; M-1 to F-313; M-1 to T-312; M-1 to E-311; M-1 to P-310; M-1 to N-309; M-1 to I-308; M-1 to G-307; M-1 to R-306; M-1 to R-305; M-1 to N-304; M-1 to Y-303; M-1 to Y-302; M-1 to G-301; M-1 to L-300; M-1 to V-299; M-1 to A-298; M-1 to V-297; M-1 to A-296; M-1 to Y-295; M-1 to R-294; M-1 to T-293; M-1 to V-292; M-1 to N-291; M-1 to D-290; M-1 to R-289; M-1 to E-288; M-1 to S-287; M-1 to Q-286; M-1 to Q-285; M-1 to I-284; M-1 to V-283; M-1 to K-282; M-1 to E-281; M-1 to L-280; M-1 to D-279; M-1 to P-278; M-1 to S-277; M-1 to D-276; M-1 toH-275; M-1 to S-274; M-1 to E-273; M-1 to G-272; M-1 to D-271; M-1 to T-270; M-1 to I-269; M-1 to V-268; M-1 to I-267; M-1 to M-266; M-1 to V-265; M-1 to K-264; M-1 to K-263; M-1 to A-262; M-1 to G-261; M-1 to K-260; M-1 to R-259; M-1 to G-258; M-1 to G-257; M-1 to K-256; M-1 to Q-255; M-1 to F-254; M-1 to A-253; M-1 to E-252; M-1 to S-251; M-1 to R-250; M-1 to A-249; M-1 to F-248; M-1 to E-247; M-1 to I-246; M-1 to G-245; M-1 to F-244; M-1 to A-243; M-1 to T-242; M-1 to R-241; M-1 to T-240; M-1 to E-239; M-1 to T-238; M-1 to G-237; M-1 to G-236; M-1 to R-235; M-1 to Q-234; M-1 to E-233; M-1 to I-232; M-1 to H-231; M-1 to S-230; M-1 to A-229; M-1 to A-228; M-1 to E-227; M-1 to V-226; M-1 to V-225; M-1 to D-224; M-1 to K-223; M-1 to V-222; M-1 to S-221; M-1 to R-220; M-1 to Y-219; M-1 to D-218; M-1 to N-217; M-1 to L-216; M-1 to H-215; M-1 to F-214; M-1 to E-213; M-1 to H-212; M-1 to V-211; M-1 to V-210; M-1 to D-209; M-1 to E-208; M-1 to G-207; M-1 to Y-206; M-1 to Q-205; M-1 to V-204; M-1 to V-203; M-1 to G-202; M-1 to V-201; M-1 to Q-200; M-1 to I-199; M-1 to Q-198; M-1 to G-197; M-1 to P-196; M-1 to G-195; M-1 to I-194; M-1 to Y-193; M-1 to F-192; M-1 to K-191; M-1 to K-190; M-1 to L-189; M-1 to I-188; M-1 to N-187; M-1 to I-186; M-1 to L-185; M-1 to F-184; M-1 to H-183; M-1 to Q-182; M-1 to V-181; M-1 to E-180; M-1 to V-179; M-1 to W-178; M-1 to P-177; M-1 to Y-176; M-1 to I-175; M-1 to S-174; M-1 to N-173; M-1 to S-172; M-1 to G-171; M-1 to D-170; M-1 to L-169; M-1 to V-168; M-1 to I-167; M-1 to V-166; M-1 to I-165; M-1 to D-164; M-1 to M-163; M-1 to Y-162; M-1 to T-161; M-1 to Q-160; M-1 to C-159; M-1 to R-158; M-1 to Q-157; M-1 to L-156; M-1 to A-155; M-1 to P-154; M-1 to A-153; M-1 to V-152; M-1 to T-151; M-1 to K-150; M-1 to S-149; M-1 to F-148; M-1 to R-147; M-1 to F-146; M-1 to N-145; M-1 to S-144; M-1 to N-143; M-1 to V-142; M-1 to R-141; M-1 to S-140; M-1 to C-139; M-1 to M-138; M-1 to G-137; M-1 to T-136; M-1 to T-135; M-1 to Y-134; M-1 to Y-133; M-1 to S-132; M-1 to S-131; M-1 to G-130; M-1 to C-129; M-1 to E-128; M-1 to H-127; M-1 to S-126; M-1 to W-125; M-1 to L-124; M-1 to P-123; M-1 to S-122; M-1 to C-121; M-1 to A-120; M-1 to L-119; M-1 to F-118; M-1 to S-117; M-1 to N-116; M-1 to D-115; M-1 to K-114; M-1 to P-113; M-1 to N-112; M-1 to T-111; M-1 to A-110; M-1 to L-109; M-1 to S-108; M-1 to L-107; M-1 to G-106; M-1 to L-105; M-1 to R-104; M-1 to M-103; M-1 to N-102; M-1 to D-101; M-1 to K-100; M-1 to R-99; M-1 to E-98; M-1 to S-97; M-1 to V-96; M-1 to N-95; M-1 to S-94; M-1 to L-93; M-1 to T-92; M-1 to V-91; M-1 to R-90; M-1 to G-89; M-1 to L-88; M-1 to N-87; M-1 to L-86; M-1 to K-85; M-1 to T-84; M-1 to C-83; M-1 to N-82; M-1 to G-81; M-1 to H-80; M-1 to I-79; M-1 to V-78; M-1 to P-77; M-1 to C-76; M-1 to K-75; M-1 to Y-74; M-1 to V-73; M-1 to D-72; M-1 to G-71; M-1 to T-70; M-1 to K-69; M-1 to Q-68; M-1 to Y-67; M-1 to G-66; M-1 to N-65; M-1 to T-64; M-1 to E-63; M-1 to L-62; M-1 to P-61; M-1 to A-60; M-1 to G-59; M-1 to V-58; M-1 to V-57; M-1 to L-56; M-1 to W-55; M-1 to K-54; M-1 to N-53; M-1 to G-52; M-1 to S-51; M-1 to I-50; M-1 to D-49; M-1 to H-48; M-1 to Q-47; M-1 to Q-46; M-1 to V-45; M-1 to T-44; M-1 to Y-43; M-1 to G-42; M-1 to F-41; M-1 to F-40; M-1 to A-39; M-1 to T-38; M-1 to R-37; M-1 to S-36; M-1 to G-35; M-1 to P-34; M-1 to I-33; M-1 to V-32; M-1 to R-31; M-1 to P-30; M-1 to K-29; M-1 to R-28; M-1 to T-27; M-1 to D-26; M-1 to M-25; M-1 to N-24; M-1 to F-23; M-1 to T-22; M-1 to D-21; M-1 to T-20; M-1 to F-19; M-1 to G-18; M-1 to P-17; M-1 to W-16; M-1 to L-15; M-1 to S-14; M-1 to L-13; M-1 to A-12; M-1 to W-11; M-1 to A-10; M-1 to V-9; M-1 to V-8; M-1 to L-7; M-1 to G-6; of SEQ ID NO:187. Polynucleotides encoding these polypeptides are also encompassed by the invention, as are antibodies that bind one or more of these polypeptides. Moreover, fragments and variants of these polypeptides (e.g., fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides and polypeptides encoded by the polynucleotide which hybridizes, under stringent conditions, to the polynucleotide encoding these polypeptides, or the complement thereof) are encompassed by the invention. Antibodies that bind these fragments and variants of the invention are also encompassed by the invention. Polynucleotides encoding these fragments and variants are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO:186 which have been determined from the following related cDNA genes: HEEAB54R (SEQ ID NO:192), HRDAF83R (SEQ ID NO:193), HOUBC62R (SEQ ID NO:194), HCDBI19R (SEQ ID NO:195), HOHCU94R (SEQ ID NO:196), HOACC13R(SEQ ID NO:197), HCDAP21R (SEQ ID NO:198) and HNHHA34R (SEQ ID NO:199), HOHEA75R (SEQ ID NO:200), and HNGEL59R (SEQ ID NO:201).

A polynucleotide encoding a polypeptide of the present invention is obtained from human ovary, small intestine, fetal heart, fetal brain, large intestine, osteoblasts, human trabelcular bone cells, messangial cells, adipocytes, osteosarcoma, chondrosarcoma, breast cancer cells, and bone marrow tissues and cells. The polynucleotide of this invention was discovered in a human osteoblast II cDNA library.

Based on the sequence similarity to the human integrin alpha 1 subunit, translation product of this gene is expected to share at least some biological activities with integrin proteins, and specifically the integrin alpha 1 protein. Such activities are known in the art, some of which are described elsewhere herein.

Specifically, polynucleotides and polypeptides of the invention, including antibodies, are also useful for modulating the differentiation of normal and malignant cells, modulating the proliferation and/or differentiation of cancer and neoplastic cells, and modulating the immune response. Polynucleotides and polypeptides of the invention may represent a diagnostic marker for hematopoietic and immune diseases and/or disorders. The full-length protein should be a secreted protein, based upon homology to the integrin family. Therefore, it is secreted into serum, urine, or feces and thus the levels is assayable from patient samples. Assuming specific expression levels are reflective of the presence of immune disorders, this protein would provide a convenient diagnostic for early detection. In addition, expression of this gene product may also be linked to the progression of immune diseases, and therefore may itself actually represent a therapeutic or therapeutic target for the treatment of cancer.

Polynucleotides and polypeptides of the invention may play an important role in the pathogenesis of human cancers and cellular transformation, particularly those of the immune and hematopoietic systems. Polynucleotides and polypeptides of the invention may also be involved in the pathogenesis of developmental abnormalities based upon its potential effects on proliferation and differentiation of cells and tissue cell types. Due to the potential proliferating and differentiating activity of said polynucleotides and polypeptides, the invention is useful as a therapeutic agent in inducing tissue regeneration, for treating inflammatory conditions (e.g., inflammatory bowel syndrome, diverticulitis, etc.). Moreover, the invention is useful in modulating the immune response to aberrant polypeptides, as may exist in rapidly proliferating cells and tissue cell types, particularly in adenocarcinoma cells, and other cancers.

Alternatively, the expression within cellular sources marked by proliferating cells indicates this protein may play a role in the regulation of cellular division, and may show utility in the diagnosis, treatment, and/or prevention of developmental diseases and disorders, including cancer, and other proliferative conditions. Representative uses are described in the “Hyperproliferative Disorders” and “Regeneration” sections below and elsewhere herein. Briefly, developmental tissues rely on decisions involving cell differentiation and/or apoptosis in pattern formation.

Dysregulation of apoptosis can result in inappropriate suppression of cell death, as occurs in the development of some cancers, or in failure to control the extent of cell death, as is believed to occur in acquired immunodeficiency and certain neurodegenerative disorders, such as spinal muscular atrophy (SMA).

Alternatively, this gene product is involved in the pattern of cellular proliferation that accompanies early embryogenesis. Thus, aberrant expression of this gene product in tissues—particularly adult tissues—may correlate with patterns of abnormal cellular proliferation, such as found in various cancers. Because of potential roles in proliferation and differentiation, this gene product may have applications in the adult for tissue regeneration and the treatment of cancers. It may also act as a morphogen to control cell and tissue type specification. Therefore, the polynucleotides and polypeptides of the present invention are useful in treating, detecting, and/or preventing said disorders and conditions, in addition to other types of degenerative conditions. Thus, this protein may modulate apoptosis or tissue differentiation and is useful in the detection, treatment, and/or prevention of degenerative or proliferative conditions and diseases. The protein is useful in modulating the immune response to aberrant polypeptides, as may exist in proliferating and cancerous cells and tissues. The protein can also be used to gain new insight into the regulation of cellular growth and proliferation. Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

This gene is expressed almost exclusively in osteoblasts, human trabelcular bone cells, messangial cells, adipocytes, and to a lesser extent in osteosarcoma, chondrosarcoma, breast cancer cells, and bone marrow.

Therefore, polynucleotides and polypeptides of the invention, including antibodies, are useful as reagents for differential identification of the tissue(s) or cell type(s) present in a biological sample and for diagnosis of the following diseases and conditions which include, but are not limited to, disorders of the skeletal system, connective tissues, and immune and hematpoietic diseases and/or disorders. Similarly, polypeptides and antibodies directed to these polypeptides are useful to provide immunological probes for differential identification of the tissue(s) or cell type(s). For a number of disorders of the above tissues or cells, particularly of the connective tissue and skeletal system, expression of this gene at significantly higher or lower levels is detected in certain tissues or cell types (e.g. immune, hematopoietic, skeletal, bone, cartilage, develpomental, reproductive, secretory, and cancerous and wounded tissues) or bodily fluids or cell types (e.g., lymph, serum, plasma, urine, synovial fluid or spinal fluid) or another tissue or cell sample taken from an individual having such a disorder, relative to the standard gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.

The tissue distribution in osteoblasts and homology to integrin alpha subunit 10 indicates that the protein products of this gene are useful for the treatment of disorders and conditions affecting the skeletal system, in particular osteoporosis as well as disorders afflicting connective tissues (e.g., arthritis, trauma, tendonitis, chrondomalacia and inflammation), such as in the diagnosis and treatment of various autoimmune disorders such as rheumatoid arthritis, lupus, scleroderma, and dermatomyositis as well as dwarfism, spinal deformation, and specific joint abnormalities as well as chondrodysplasias (i.e., spondyloepiphyseal dysplasia congenita, familial osteoarthritis, Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid). polynucleotides and polypeptides corresponding to this gene are useful for the treatment and diagnosis of hematopoietic related disorders such as anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. Such a use is consistent with the observed homology to integrin family members, in conjunction with the tissue distribution in bone marrow cells.

Integrins play pivotal roles in cell migration, inflammation, proliferation, and cellular infiltration. Thus, the present invention is expected to share at least some of these activities. Representative uses are described in the “Immune Activity” and “infectious disease” sections below, in Examples 17, 42, 44, 45, 47, 49, 50, and 51, and elsewhere herein. Briefly, the uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia. The gene product may also be involved in lymphopoiesis, therefore, it can be used in immune disorders such as infection, inflammation, allergy, immunodeficiency etc. In addition, this gene product may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types. Based upon the tissue distribution of this protein, antagonists directed against this protein is useful in blocking the activity of this protein. Accordingly, preferred are antibodies which specifically bind a portion of the translation product of this gene.

Also provided is a kit for detecting tumors in which expression of this protein occurs. Such a kit comprises in one embodiment an antibody specific for the translation product of this gene bound to a solid support. Also provided is a method of detecting these tumors in an individual which comprises a step of contacting an antibody specific for the translation product of this gene to a bodily fluid from the individual, preferably serum, and ascertaining whether antibody binds to an antigen found in the bodily fluid. Preferably the antibody is bound to a solid support and the bodily fluid is serum. The above embodiments, as well as other treatments and diagnostic tests (kits and methods), are more particularly described elsewhere herein.

Furthermore, the protein may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate cognate ligands or receptors, to identify agents that modulate their interactions, in addition to its use as a nutritional supplement. Protein, as well as, antibodies directed against the protein may show utility as a tumor marker and/or immunotherapy targets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases. Some of these sequences are related to SEQ ID NO:186 and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence is cumbersome. Accordingly, preferably excluded from the present invention are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 4981 of SEQ ID NO:186, b is an integer of 15 to 4995, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:186, and where b is greater than or equal to a+14.

Tables Description of Table 1A

Table 1A summarizes information concerning certain polynucleotides and polypeptides of the invention. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, “cDNA Clone ID”, for a cDNA clone related to each contig sequence disclosed in Table 1A. The cDNA Clones identified in the second column were deposited as indicated in the third column (i.e. by ATCC™ Deposit No:Z and deposit date). Some of the deposits contain multiple different clones corresponding to the same gene. In the fourth column, “Vector” refers to the type of vector contained in the corresponding cDNA Clone identified in the second column. In the fifth column, the nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the corresponding cDNA clone identified in the second column and, in some cases, from additional related cDNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X. In the sixth column, “Total NT Seq.” refers to the total number of nucleotides in the contig sequence identified as SEQ ID NO:X.” The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5′ NT of Clone Seq.” (seventh column) and the “3′ NT of Clone Seq.” (eighth column) of SEQ ID NO:X. In the ninth column, the nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon.” Similarly, in column ten, the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as “5′ NT of First AA of Signal Pep.” In the eleventh column, the translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be routinely translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

In the twelfth and thirteenth columns of Table 1A, the first and last amino acid position of SEQ ID NO:Y of the predicted signal peptide is identified as “First AA of Sig Pep” and “Last AA of Sig Pep.” In the fourteenth column, the predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as “Predicted First AA of Secreted Portion”. The amino acid position of SEQ ID NO:Y of the last amino acid encoded by the open reading frame is identified in the fifteenth column as “Last AA of ORF”.

SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1A and/or elsewhere herein.

Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).

Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC™, as set forth in Table 1A. The nucleotide sequence of each deposited plasmid can readily be determined by sequencing the deposited plasmid in accordance with known methods

The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular plasmid can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.

Also provided in Table 1A is the name of the vector which contains the cDNA plasmid. Each vector is routinely used in the art. The following additional information is provided for convenience.

Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBLUESCRIPT™ (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from STRATAGENE™ Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from STRATAGENE™.

Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from LIFE TECHNOLOGIES™, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from LIFE TECHNOLOGIES™. See, for instance, Gruber, C. E., et al., Focus 15:59 (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from LIFE TECHNOLOGIES™. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).

The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or a deposited cDNA (cDNA Clone ID). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include, but are not limited to, preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X and SEQ ID NO:Y using information from the sequences disclosed herein or the clones deposited with the ATCC™. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X and/or a cDNA contained in ATCC™ Deposit No:Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, and/or a polypeptide encoded by a cDNA contained in ATCC™ Deposit No:Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X and/or a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z, are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the complement of the coding strand of the cDNA contained in ATCC™ Deposit No:Z.

Description of Tables 1B.1 and 1B.2

Tables 1B.1 and 1B.2 summarize some of the polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID:), contig sequences (contig identifier (Contig ID:) and contig nucleotide sequence identifiers (SEQ ID NO:X)) and further summarizes certain characteristics of these polynucleotides and the polypeptides encoded thereby. The first column of Tables 1B.1 and 1B.2 provide the gene numbers in the application for each clone identifier. The second column of Tables 1B.1 and 1B.2 provide unique clone identifiers, “cDNA Clone ID”, for cDNA clones related to each contig sequence disclosed in Table 1A and/or Tables 1B.1 and 1B.2. The third column of Tables 1B.1 and 1B.2 provide unique contig identifiers, “Contig ID:” for each of the contig sequences disclosed in these tables. The fourth column of Tables 1B.1 and 1B.2 provide the sequence identifiers, “SEQ ID NO:X”, for each of the contig sequences disclosed in Table 1A and/or Tables 1B.1 and 1B.2. Table 1B.1

The fifth column of Table 1B.1, “ORF (From-To)”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:X that delineates the preferred open reading frame (ORF) that encodes the amino acid sequence shown in the sequence listing and referenced in Table 1B.1 as SEQ ID NO:Y (column 6). Column 7 of Table 1B.1 lists residues comprising predicted epitopes contained in the polypeptides encoded by each of the preferred ORFs (SEQ ID NO:Y). Identification of potential immunogenic regions was performed according to the method of Jameson and Wolf (CABIOS, 4; 181-186 (1988)); specifically, the Genetics Computer Group (GCG) implementation of this algorithm, embodied in the program PEPTIDESTRUCTURE (Wisconsin Package v10.0, Genetics Computer Group (GCG), Madison, Wis.). This method returns a measure of the probability that a given residue is found on the surface of the protein. Regions where the antigenic index score is greater than 0.9 over at least 6 amino acids are indicated in Table 1B.1 as “Predicted Epitopes”. In particular embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the predicted epitopes described in Table 1B.1. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly. Column 8 of Table 1B.1 (“Cytologic Band”) provides the chromosomal location of polynucleotides corresponding to SEQ ID NO:X. Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Information) UniGene database. Given a presumptive chromosomal location, disease locus association was determined by comparison with the Morbid Map, derived from OMIM™ (“Online Mendelian Inheritance in Man,” McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.) 2000 (world wide web at ncbi.nlm.nih.gov/omim/). If the putative chromosomal location of the Query overlaps with the chromosomal location of a Morbid Map entry, an OMIM identification number is disclosed in Table 1B.1, column 9 labeled “OMIM Disease Reference(s)”. Table 5 is a key to the OMIM reference identification numbers (Table 5, column 1), and provides a description of the associated disease in Table 5, column 2. Table 1B.2

Column 5 of Table 1B.2, “Tissue Distribution” shows the expression profile of tissue, cells, and/or cell line libraries which express the polynucleotides of the invention. The first code number shown in Table 1B.2 column 5 (preceding the colon), represents the tissue/cell source identifier code corresponding to the key provided in Table 4. Expression of these polynucleotides was not observed in the other tissues and/or cell libraries tested. The second number in column 5 (following the colon), represents the number of times a sequence corresponding to the reference polynucleotide sequence (e.g., SEQ ID NO:X) was identified in the corresponding tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are “AR” designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of ³³P dCTP, using oligo(dT) to prime reverse transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after “[array code]:” represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased tissue(s) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression.

Description of Table 1C

Table 1C summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID:), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ ID NO:B). The first column provides a unique clone identifier, “Clone ID:”, for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, “SEQ ID NO:X”, for each contig sequence. The third column provides a unique contig identifier, “Contig ID:” for each contig sequence. The fourth column, provides a BAC identifier “BAC ID NO:A” for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, “SEQ ID NO:B” for a fragment of the BAC clone identified in column four of the corresponding row of the table. The sixth column, “Exon From-To”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof). Table 1C in priority Application No. PCT/US02/09785, filed Mar. 19, 2002, which corresponds to Publication No. WO02/95010, published Nov. 28, 2002 (e.g., pages 228 to 235 of Publication No. WO02/95010) is incorporated by reference herein in its entirety.

Description of Tables 1D, 1E, 1E.1 and 1E.2

Table 1D: In preferred embodiments, the present invention encompasses a method of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder listed as listed in the “Preferred Indications” column of Table 1D, column 3; comprising administering to a patient (in which such detection, prevention, treatment, and/or amelioration is desired) a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) represented by, for example, Table 1A or 1D (in the same row as the disease or disorder to be treated is listed in the “Preferred Indications” column of Tables 1D) in an amount effective to detect, prevent, diagnose, prognosticate, treat, or ameliorate the disease or disorder. In preferred embodiments, the present invention encompasses a method of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder (such as an immune, cardiovascular, cancer, or other proliferative disease or disorder), comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) represented by Tables 1A, 1B.1, 1B.2, and 1C, in an amount effective to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate the disease or disorder.

As indicated in Table 1D, the polynucleotides, polypeptides, agonists, or antagonists of the present invention (including antibodies) can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists thereof (including antibodies) could be used to prevent, treat, or ameliorate the associated disease.

The present invention encompasses methods of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of detecting, diagnosing, treating, preventing, or ameliorating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to treat, prevent, diagnose, or ameliorate the disease or disorder. The first and second columns of Table 1D show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in Column 3 of Table 1D.

In another embodiment, the present invention also encompasses methods of preventing, treating, diagnosing, or ameliorating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in Column 3 of Table 1D.

The “Preferred Indication” column describes diseases, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).

The recitation of “Cancer” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., leukemias, cancers, and/or as described below under “Hyperproliferative Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D may be used for example, to diagnose, treat, prevent, and/or ameliorate a neoplasm located in a tissue selected from the group consisting of: colon, abdomen, bone, breast, digestive system, liver, pancreas, prostate, peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a pre-neoplastic condition, selected from the group consisting of: hyperplasia (e.g., endometrial hyperplasia and/or as described in the section entitled “Hyperproliferative Disorders”), metaplasia (e.g., connective tissue metaplasia, atypical metaplasia, and/or as described in the section entitled “Hyperproliferative Disorders”), and/or dysplasia (e.g., cervical dysplasia, and bronchopulmonary dysplasia).

In another specific embodiment, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a benign dysproliferative disorder selected from the group consisting of: benign tumors, fibrocystic conditions, tissue hypertrophy, and/or as described in the section entitled “Hyperproliferative Disorders”.

The recitation of “Immune/Hematopoietic” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having the “Immune/Hematopoietic” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: anemia, pancytopenia, leukopenia, thrombocytopenia, leukemias, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis, granulomatous disease, immune deficiency, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, systemic lupus erythematosis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and allergies.

The recitation of “Reproductive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the reproductive system (e.g., as described below under “Reproductive System Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Reproductive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cryptorchism, prostatitis, inguinal hernia, varicocele, leydig cell tumors, verrucous carcinoma, prostatitis, malacoplakia, Peyronie's disease, penile carcinoma, squamous cell hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner's syndrome, mucopurulent cervicitis, Sertoli-leydig tumors, ovarian cancer, uterine cancer, pelvic inflammatory disease, testicular cancer, prostate cancer, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, testicular atrophy, testicular feminization, anorchia, ectopic testis, epididymitis, orchitis, gonorrhea, syphilis, testicular torsion, vasitis nodosa, germ cell tumors, stromal tumors, dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding, cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, cervical neoplasms, pseudohermaphroditism, and premenstrual syndrome.

The recitation of “Musculoskeletal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the immune system (e.g., as described below under “Immune Activity”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Musculoskeletal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bone cancers (e.g., osteochondromas, benign chondromas, chondroblastoma, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, multiple myeloma, osteosarcomas), Paget's Disease, rheumatoid arthritis, systemic lupus erythematosus, osteomyelitis, Lyme Disease, gout, bursitis, tendonitis, osteoporosis, osteoarthritis, muscular dystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis.

The recitation of “Cardiovascular” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cardiovascular” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: myxomas, fibromas, rhabdomyomas, cardiovascular abnormalities (e.g., congenital heart defects, cerebral arteriovenous malformations, septal defects), heart disease (e.g., heart failure, congestive heart disease, arrhythmia, tachycardia, fibrillation, pericardial Disease, endocarditis), cardiac arrest, heart valve disease (e.g., stenosis, regurgitation, prolapse), vascular disease (e.g., hypertension, coronary artery disease, angina, aneurysm, arteriosclerosis, peripheral vascular disease), hyponatremia, hypernatremia, hypokalemia, and hyperkalemia.

The recitation of “Mixed Fetal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Mixed Fetal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: spina bifida, hydranencephaly, neurofibromatosis, fetal alcohol syndrome, diabetes mellitus, PKU, Down's syndrome, Patau syndrome, Edwards syndrome, Turner syndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crouzon syndrome, cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel-Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome, Smith-Lemli-Opitz syndrome, thromocytopenia-absent radius (TAR) syndrome, Treacher Collins syndrome, Williams syndrome, Hirschsprung's disease, Meckel's diverticulum, polycystic kidney disease, Turner's syndrome, and gonadal dysgenesis, Klippel-Feil syndrome, Ostogenesis imperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumor, neuroblastoma, and retinoblastoma.

The recitation of “Excretory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and renal disorders (e.g., as described below under “Renal Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Excretory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bladder cancer, prostate cancer, benign prostatic hyperplasia, bladder disorders (e.g., urinary incontinence, urinary retention, urinary obstruction, urinary tract Infections, interstitial cystitis, prostatitis, neurogenic bladder, hematuria), renal disorders (e.g., hydronephrosis, proteinuria, renal failure, pyelonephritis, urolithiasis, reflux nephropathy, and unilateral obstructive uropathy).

The recitation of “Neural/Sensory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the nervous system (e.g., as described below under “Neural Activity and Neurological Diseases”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Neural/Sensory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: brain cancer (e.g., brain stem glioma, brain tumors, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, and cerebral astrocytoma, neurodegenerative disorders (e.g., Alzheimer's Disease, Creutzfeldt-Jakob Disease, Parkinson's Disease, and Idiopathic Presenile Dementia), encephalomyelitis, cerebral malaria, meningitis, metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylase deficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS Dementia Complex, schizophrenia, attention deficit disorder, hyperactive attention deficit disorder, autism, and obsessive compulsive disorders.

The recitation of “Respiratory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Respiratory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of the respiratory system such as larynx cancer, pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamous cell carcinomas, small cell (oat cell) carcinomas, large cell carcinomas, and adenocarcinomas. Allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X, infiltrative lung diseases (e.g., pulmonary fibrosis and lymphoid interstitial pneumonia), obstructive airway diseases (e.g., asthma, emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis and asbestosis), pneumonia, and pleurisy.

The recitation of “Endocrine” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”), renal disorders (e.g., as described below under “Renal Disorders”), and disorders of the endocrine system (e.g., as described below under “Endocrine Disorders”.

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having an “Endocrine” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of endocrine tissues and organs (e.g., cancers of the hypothalamus, pituitary gland, thyroid gland, parathyroid glands, pancreas, adrenal glands, ovaries, and testes), diabetes (e.g., diabetes insipidus, type I and type II diabetes mellitus), obesity, disorders related to pituitary glands (e.g., hyperpituitarism, hypopituitarism, and pituitary dwarfism), hypothyroidism, hyperthyroidism, goiter, reproductive disorders (e.g. male and female infertility), disorders related to adrenal glands (e.g., Addison's Disease, corticosteroid deficiency, and Cushing's Syndrome), kidney cancer (e.g., hypernephroma, transitional cell cancer, and Wilm's tumor), diabetic nephropathy, interstitial nephritis, polycystic kidney disease, glomerulonephritis (e.g., IgM mesangial proliferative glomerulonephritis and glomerulonephritis caused by autoimmune disorders; such as Goodpasture's syndrome), and nephrocalcinosis.

The recitation of “Digestive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the gastrointestinal system (e.g., as described below under “Gastrointestinal Disorders”.

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Digestive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portal hypertension, cholelithiasis, cancer of the digestive system (e.g., biliary tract cancer, stomach cancer, colon cancer, gastric cancer, pancreatic cancer, cancer of the bile duct, tumors of the colon (e.g., polyps or cancers), and cirrhosis), pancreatitis, ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atropy, benign tumors of the duodenum, distension, irritable bowel syndrome, malabsorption, congenital disorders of the small intestine, bacterial and parasitic infection, megacolon, Hirschsprung's disease, aganglionic megacolon, acquired megacolon, colitis, anorectal disorders (e.g., anal fistulas, hemorrhoids), congenital disorders of the liver (e.g., Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and alpha1-antitrypsin deficiency), portal hypertension, cholelithiasis, and jaundice.

The recitation of “Connective/Epithelial” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), and or to promote or inhibit regeneration (e.g., as described below under “Regeneration”), and wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Connective/Epithelial” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: connective tissue metaplasia, mixed connective tissue disease, focal epithelial hyperplasia, epithelial metaplasia, mucoepithelial dysplasia, graft v. host disease, polymyositis, cystic hyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer's disease, lymphoproliferative disorder, Waldenstron's macroglobulinemia, Crohn's disease, pernicious anemia, idiopathic Addison's disease, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease, wound healing, relapsing polychondritis, vasculitis, polyarteritis nodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderma, CREST syndrome, Sjogren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease, relapsing polychondritis, vasculitis, Henoch-Schonlein syndrome, erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis, Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, cellulitis, keloids, Ehler Danlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogenese imperfecta, chondrodysplasias, epidermolysis bullosa, Alport syndrome, and cutis laxa.

Table 1E provides information related to biological activities and preferred indications for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1E also provides information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“cDNA Clone ID:”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B.1, 1B.2, 1C, and 1D. The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A and 1B.1). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The sixth column (“Preferred Indications”) describes particular embodiments of the invention and indications (e.g. pathologies, diseases, disorders, abnormalities, etc.) for which polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) may be used in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating.

Tables 1E.1 and 1E.2 provide information related to biological activities for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Tables 1E.2 also provide information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column of Table 1E.1 (“Gene No.”) provides the gene number in the application for each clone identifier. The second column of Table 1E.1 (“cDNA Clone ID:”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B.1, 1B.2, and 1C. The third column of Table 1E.1 (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A and 1B.1). The fourth column of Table 1E.1 (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides).

In Table 1E.2, each of the biological activities of Table 1E.1 are listed by “Biological Activity Number” and the corresponding “Biological Activity” and are followed by an “Exemplary Activity Assay” column and a “Preferred Indication” column; however, for some biological activities no “Exemplary Activity Assay” or “Preferred Indication” is given. The “Exemplary Activity Assay” column describes the biological activity listed in the column that precedes it and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The “Preferred Indication” column also refers to the biological activity listed in the preceding column and describes disease(s) or disorder(s) that may be detected, diagnosed, prevented, treated, or ameliorated by the nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof).

Tables 1E, 1E.1, and 1E.2 describe the use of FMAT technology, inter alia, for testing or demonstrating various biological activities. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based system that provides a means to perform nonradioactive cell- and bead-based assays to detect activation of cell signal transduction pathways. This technology was designed specifically for ligand binding and immunological assays. Using this technology, fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using a data processing system. Unbound fluorophore comprising the background signal is ignored, allowing for a wide variety of homogeneous assays. FMAT technology may be used for peptide ligand binding assays, immunofluorescence, apoptosis, cytotoxicity, and bead-based immunocapture assays. See, Miraglia S et. al., “Homogeneous cell and bead based assays for high throughput screening using fluorometric microvolume assay technology,” Journal of Biomolecular Screening; 4:193-204 (1999). In particular, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides (including polypeptide fragments and variants) to activate signal transduction pathways. For example, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides to upregulate production of immunomodulatory proteins (such as, for example, interleukins, GM-CSF, Rantes, and Tumor Necrosis factors, as well as other cellular regulators (e.g. insulin)).

Tables 1E, 1E.1, and 1E.2 also describe the use of kinase assays for testing, demonstrating, or quantifying biological activity. In this regard, the phosphorylation and de-phosphorylation of specific amino acid residues (e.g. Tyrosine, Serine, Threonine) on cell-signal transduction proteins provides a fast, reversible means for activation and de-activation of cellular signal transduction pathways. Moreover, cell signal transduction via phosphorylation/de-phosphorylation is crucial to the regulation of a wide variety of cellular processes (e.g. proliferation, differentiation, migration, apoptosis, etc.). Accordingly, kinase assays provide a powerful tool useful for testing, confirming, and/or identifying polypeptides (including polypeptide fragments and variants) that mediate cell signal transduction events via protein phosphorylation. See e.g., Forrer, P., Tamaskovic R., and Jaussi, R. “Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and p38 Kinase Activities” Biol. Chem. 379(8-9): 1101-1110 (1998).

Description of Table 1F

Polynucleotides encoding polypeptides of the present invention can be used in assays to test for one or more biological activities. One such biological activity which may be tested includes the ability of polynucleotides and polypeptides of the invention to stimulate up-regulation or down-regulation of expression of particular genes and proteins. Hence, if polynucleotides and polypeptides of the present invention exhibit activity in altering particular gene and protein expression patterns, it is likely that these polynucleotides and polypeptides of the present invention may be involved in, or capable of effecting changes in, diseases associated with the altered gene and protein expression profiles. Hence, polynucleotides, polypeptides, or antibodies of the present invention could be used to treat said associated diseases.

TaqMan® assays may be performed to assess the ability of polynucleotides (and polypeptides they encode) to alter the expression pattern of particular “target” genes. TaqMan® reactions are performed to evaluate the ability of a test agent to induce or repress expression of specific genes in different cell types. TaqMan® gene expression quantification assays (“TaqMan® assays”) are well known to, and routinely performed by, those of ordinary skill in the art. TaqMan® assays are performed in a two step reverse transcription/polymerase chain reaction (RT-PCR). In the first (RT) step, cDNA is reverse transcribed from total RNA samples using random hexamer primers. In the second (PCR) step, PCR products are synthesized from the cDNA using gene specific primers.

To quantify gene expression the TaqMan® PCR reaction exploits the 5′ nuclease activity of AmpliTaq Gold® DNA Polymerase to cleave a TaqMan® probe (distinct from the primers) during PCR. The TaqMan® probe contains a reporter dye at the 5′-end of the probe and a quencher dye at the 3′ end of the probe. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. AmpliTaq Gold® DNA Polymerase then cleaves the probe between the reporter and quencher when the probe hybridizes to the target, resulting in increased fluorescence of the reporter (see FIG. 2). Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye.

After the probe fragments are displaced from the target, polymerization of the strand continues. The 3′-end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. Because of these requirements, any nonspecific amplification is not detected.

For test sample preparation, vector controls or constructs containing the coding sequence for the gene of interest are transfected into cells, such as for example 293T cells, and supernatants collected after 48 hours. For cell treatment and RNA isolation, multiple primary human cells or human cell lines are used; such cells may include but are not limited to, Normal Human Dermal Fibroblasts, Aortic Smooth Muscle, Human Umbilical Vein Endothelial Cells, HepG2, Daudi, Jurkat, U937, Caco, and THP-1 cell lines. Cells are plated in growth media and growth is arrested by culturing without media change for 3 days, or by switching cells to low serum media and incubating overnight. Cells are treated for 1, 6, or 24 hours with either vector control supernatant or sample supernatant (or purified/partially purified protein preparations in buffer). Total RNA is isolated; for example, by using TRIZOL™ extraction or by using the Ambion RNAqueous™-4PCR RNA isolation system. Expression levels of multiple genes are analyzed using TaqMan®, and expression in the test sample is compared to control vector samples to identify genes induced or repressed. Each of the above described techniques are well known to, and routinely performed by, those of ordinary skill in the art.

Table 1F indicates particular disease classes and preferred indications for which polynucleotides, polypeptides, or antibodies of the present invention may be used in detecting, diagnosing, preventing, treating and/or ameliorating said diseases and disorders based on “target” gene expression patterns which may be up- or down-regulated by polynucleotides (and the encoded polypeptides) corresponding to each indicated cDNA Clone ID (shown in Table 1F, Column 2).

Thus, in preferred embodiments, the present invention encompasses a method of detecting, diagnosing, preventing, treating, and/or ameliorating a disease or disorder listed in the “Disease Class” and/or “Preferred Indication” columns of Table 1F; comprising administering to a patient in which such detection, diagnosis, prevention, or treatment is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to detect, diagnose, prevent, treat, or ameliorate the disease or disorder. The first and second columns of Table 1F show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in detecting, diagnosing, preventing, treating, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in the “Disease Class” or “Preferred Indication” Columns of Table 1F.

In another embodiment, the present invention also encompasses methods of detecting, diagnosing, preventing, treating, or ameliorating a disease or disorder listed in the “Disease Class” or “Preferred Indication” Columns of Table 1F; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in the “Disease Class” or “Preferred Indication” Columns of Table 1F.

The “Disease Class” Column of Table 1F provides a categorized descriptive heading for diseases, disorders, and/or conditions (more fully described below) that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).

The “Preferred Indication” Column of Table 1F describes diseases, disorders, and/or conditions that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).

The “Cell Line” and “Exemplary Targets” Columns of Table 1F indicate particular cell lines and target genes, respectively, which may show altered gene expression patterns (i.e., up- or down-regulation of the indicated target gene) in TaqMan® assays, performed as described above, utilizing polynucleotides of the cDNA Clone ID shown in the corresponding row. Alteration of expression patterns of the indicated “Exemplary Target” genes is correlated with a particular “Disease Class” and/or “Preferred Indication” as shown in the corresponding row under the respective column headings.

The “Exemplary Accessions” Column indicates GenBank Accessions (available online through the National Center for Biotechnology Information (NCBI) at the world wide web at ncbi.nlm.nih.gov/) which correspond to the “Exemplary Targets” shown in the adjacent row.

The recitation of “Cancer” in the “Disease Class” Column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate neoplastic diseases and/or disorders (e.g., leukemias, cancers, etc., as described below under “Hyperproliferative Disorders”).

The recitation of “Immune” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).

The recitation of “Angiogenesis” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), diseases and/or disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”), diseases and/or disorders involving cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), diseases and/or disorders involving angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), to promote or inhibit cell or tissue regeneration (e.g., as described below under “Regeneration”), or to promote wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”). Highly preferred indications include diagnosis, prevention, treatment, and/or amelioration of diseases and disorders involving angiogenesis, wound healing, neoplasia (particularly including, but not limited to, tumor metastases), and cardiovascular diseases and disorders; as described herein under the headings “Hyperproliferative Disorders,” “Regeneration,” “Anti-Angiogenesis Activity,” “Diseases at the Cellular Level,” and “Wound Healing and Epithelial Cell Proliferation.”

The recitation of “Diabetes” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diabetes (including diabetes mellitus types I and II), as well as diseases and/or disorders associated with, or consequential to, diabetes (e.g. as described below under “Endocrine Disorders,” “Renal Disorders,” and “Gastrointestinal Disorders”).

Description of Table 2

Table 2 summarizes homology and features of some of the polypeptides of the invention. The first column provides a unique clone identifier, “Clone ID:”, corresponding to a cDNA clone disclosed in Tables 1A, 1B.1, and/or 1B.2. The second column provides the unique contig identifier, “Contig ID:” corresponding to contigs in Tables 1B.1 and 1B.2 and allowing for correlation with the information in Tables 1B.1 and 1B.2. The third column provides the sequence identifier, “SEQ ID NO:X”, for the contig polynucleotide sequence. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. Comparisons were made between polypeptides encoded by the polynucleotides of the invention and either a non-redundant protein database (herein referred to as “NR”), or a database of protein families (herein referred to as “PFAM”) as further described below. The fifth column provides a description of the PFAM/NR hit having a significant match to a polypeptide of the invention. Column six provides the accession number of the PFAM/NR hit disclosed in the fifth column. Column seven, “Score/Percent Identity”, provides a quality score or the percent identity, of the hit disclosed in columns five and six. Columns 8 and 9, “NT From” and “NT To” respectively, delineate the polynucleotides in “SEQ ID NO:X” that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth and sixth columns. In specific embodiments polypeptides of the invention comprise, or alternatively consist of, an amino acid sequence encoded by a polynucleotide in SEQ ID NO:X as delineated in columns 8 and 9, or fragments or variants thereof.

Description of Table 3

Table 3 provides polynucleotide sequences that may be disclaimed according to certain embodiments of the invention. The first column provides a unique clone identifier, “cDNA Clone ID”, for a cDNA clone related to contig sequences disclosed in Tables 1B.1 and 1B.2. The second column provides the sequence identifier, “SEQ ID NO:X”, for contig sequences disclosed in Tables 1A, 1B.1, and/or 1B.2. The third column provides the unique contig identifier, “Contig ID:”, for contigs disclosed in Tables 1B.1 and 1B.2. In specific embodiments of the invention, for each “Contig ID” listed in the third column of Table 3, preferably excluded are one or more polynucleotides comprising, or alternatively consisting of, SEQ ID NO:X referenced in the second column of Table 3 and described by the general formula of a-b, whereas a and b are uniquely defined integers determined for the corresponding SEQ ID NO:X referred to in column 2 of Table 3. The fourth column provides a range of values for the unique integer ‘a’ where ‘a’ is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, and the fifth column provides a range of values for the unique integer ‘b’ where ‘b’ is any integer between 15 and the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a+14. In certain embodiments, preferably excluded from the invention are at least one, two, three, four, or more of the polynucleotide sequence(s) having the accession number(s) disclosed in the sixth column of Table 3 (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are at least five, ten, or more of the polynucleotide sequence(s) having the accession number(s) disclosed in the sixth column of this Table (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least one, two, three, four, or more of the available material having the accession numbers identified in the sixth column of Table 3 (including for example, the actual sequence contained in an identified BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least five, ten, or more of the available material having the accession numbers identified in the sixth column of Table 3 (including for example, the actual sequence contained in an identified BAC clone). In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety.

Description of Table 4

Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1B.2, column 5. Column 1 provides the tissue/cell source identifier code disclosed in Table 1B.2, column 5. Columns 2-5 provide a description of the tissue or cell source. Note that “Description” and “Tissue” sources (i.e. columns 2 and 3) having the prefix “a_” indicates organs, tissues, or cells derived from “adult” sources. Codes corresponding to diseased tissues are indicated in column 6 with the word “disease.” The use of the word “disease” in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the “disease” designation may still be derived from sources directly or indirectly involved in a disease state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where the tissue/cell source is a library, column 7 identifies the vector used to generate the library.

Description of Table 5

Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1B.1, column 9. OMIM reference identification numbers (column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, Md.) 2000 (world wide web at ncbi.nlm.nih.gov/omim/). Column 2 provides diseases associated with the cytologic band disclosed in Table 1B.1, column 8 as determined using the Morbid Map database.

Description of Table 6

Table 6 summarizes some of the ATCC™ Deposits, Deposit dates, and ATCC™ designation numbers of deposits made with the ATCC™ in connection with the present application. These deposits were made in addition to those described in Table 1A.

Description of Table 7

Table 7 shows the cDNA libraries sequenced, and ATCC™ designation numbers and vector information relating to these cDNA libraries.

The first column shows the first four letters indicating the Library from which each library clone was derived. The second column indicates the catalogued tissue description for the corresponding libraries. The third column indicates the vector containing the corresponding clones. The fourth column shows the ATCC™ deposit designation for each library clone as indicated by the deposit information in Table 6.

DEFINITIONS

The following definitions are provided to facilitate understanding of certain terms used throughout this specification.

In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. The term “isolated” does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.

In the present invention, a “secreted” protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a “mature” protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.

As used herein, a “polynucleotide” refers to a molecule having a nucleic acid sequence encoding SEQ ID NO:Y or a fragment or variant thereof (e.g., the polypeptide delineated in columns fourteen and fifteen of Table 1A); a nucleic acid sequence contained in SEQ ID NO:X (as described in column 5 of Table 1A and/or column 4 of Tables 1B.1 and 1B.2) or the complement thereof a cDNA sequence contained in Clone ID: (as described in column 2 of Table 1A and/or Tables 1B.1 and 1B.2 and contained within a library deposited with the ATCC™); a nucleotide sequence encoding the polypeptide encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 (EXON From-To) of Table 1C or a fragment or variant thereof or a nucleotide coding sequence in SEQ ID NO:B as defined in column 6 of Table 1C or the complement thereof. For example, the polynucleotide can contain the nucleotide sequence of the full length cDNA sequence, including the 5′ and 3′ untranslated sequences, the coding region (with or without a natural or artificial signal sequence), the protein coding region as well as fragments, epitopes, domains, and variants of the nucleic acid sequence. Moreover, as used herein, a “polypeptide” refers to a molecule having an amino acid sequence encoded by a polynucleotide of the invention as broadly defined (obviously excluding poly-Phenylalanine or poly-Lysine peptide sequences which result from translation of a polyA tail of a sequence corresponding to a cDNA).

In the present invention, “SEQ ID NO:X” was often generated by overlapping sequences contained in multiple clones (contig analysis). A representative clone containing all or most of the sequence for SEQ ID NO:X is deposited at Human Genome Sciences, Inc. (HGS) in a catalogued and archived library. As shown, for example, in column 2 of Tables 1B.1 and 1B.2, each clone is identified by a cDNA Clone ID (identifier generally referred to herein as Clone ID:). Each Clone ID is unique to an individual clone and the Clone ID is all the information needed to retrieve a given clone from the HGS library. Table 7 provides a list of the deposited cDNA libraries. One can use the Clone ID: to determine the library source by reference to Tables 6 and 7. Table 7 lists the deposited cDNA libraries by name and links each library to an ATCC™ Deposit. Library names contain four characters, for example, “HTWE.” The name of a cDNA clone (Clone ID) isolated from that library begins with the same four characters, for example “HTWEP07”. As mentioned below, Tables 1A, 1B.1 and 1B.2 correlate the Clone ID names with SEQ ID NO:X. Thus, starting with an SEQ ID NO:X, one can use Tables 1A, 1B.1, 1B.2, 6, and 7 to determine the corresponding Clone ID, which library it came from and which ATCC™ deposit the library is contained in. Furthermore, it is possible to retrieve a given cDNA clone from the source library by techniques known in the art and described elsewhere herein. The ATCC™ is located at 10801 University Boulevard, Manassas, Va. 20110-2209, USA. The ATCC™ deposits were made pursuant to the terms of the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure.

In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

A “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NO:X, or the complement thereof (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments described herein), the polynucleotide sequence delineated in columns 7 and 8 of Table 1A or the complement thereof, the polynucleotide sequence delineated in columns 8 and 9 of Table 2 or the complement thereof, and/or cDNA sequences contained in Clone ID: (e.g., the complement of any one, two, three, four, or more of the polynucleotide fragments, or the cDNA clone within the pool of cDNA clones deposited with the ATCC™, described herein), and/or the polynucleotide sequence delineated in column 6 of Table 1C or the complement thereof. “Stringent hybridization conditions” refers to an overnight incubation at 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37 degree C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA; followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences (such as any 3′ terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of “polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.

In specific embodiments, the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron. In another embodiment, the polynucleotides comprising coding sequences do not contain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ to the gene of interest in the genome). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).

The polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

“SEQ ID NO:X” refers to a polynucleotide sequence described, for example, in Tables 1A, 1B.1, 1B.2, 1C, 2, and/or 3, while “SEQ ID NO:Y” refers to a polypeptide sequence described in, for example, column 11 of Table 1A; column 6 of Table 1B.1; and/or column 3 of Table 1E or 1E.1. SEQ ID NO:X is identified by an integer specified in, for example, column 5 of Table 1A; column 4 of Table 1B.1 or 1B.2; column 2 of Table 1C; column 3 of Table 2; and/or column 2 of Table 3. The polypeptide sequence SEQ ID NO:Y is a translated open reading frame (ORF) encoded by polynucleotide SEQ ID NO:X. “Clone ID:” refers to a cDNA clone described in, for example, column 2 of Tables 1A, 1B.1, 1B.2, 1D, 1E, 1E.1, and/or 1F; and/or column 1 of Table 1C, 2, and/or 3.

“A polypeptide having functional activity” refers to a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein. Such functional activities include, but are not limited to, biological activity (e.g. activity useful in treating, preventing and/or ameliorating diseases and disorders such as immune, cardiovascular, cancer, and other proliferative diseases and disorders), antigenicity (ability to bind [or compete with a polypeptide for binding] to an anti-polypeptide antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide.

The polypeptides of the invention can be assayed for functional activity (e.g. biological activity) using or routinely modifying assays known in the art, as well as assays described herein. Specifically, one of skill in the art may routinely assay secreted polypeptides (including fragments, variants, derivatives, and analogs) of the invention for activity using assays as described in the examples section below.

“A polypeptide having biological activity” refers to a polypeptide exhibiting activity similar to, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about tenfold less activity, and most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).

Tables Table 1A

Table 1A summarizes information concerning certain polynucleotides and polypeptides of the invention. The first column provides the gene number in the application for each clone identifier. The second column provides a unique clone identifier, “Clone ID:”, for a cDNA clone related to each contig sequence disclosed in Table 1A. Third column, the cDNA Clones identified in the second column were deposited as indicated in the third column (i.e. by ATCC™ Deposit No:Z and deposit date). Some of the deposits contain multiple different clones corresponding to the same gene. In the fourth column, “Vector” refers to the type of vector contained in the corresponding cDNA Clone identified in the second column. In the fifth column, the nucleotide sequence identified as “NT SEQ ID NO:X” was assembled from partially homologous (“overlapping”) sequences obtained from the corresponding cDNA clone identified in the second column and, in some cases, from additional related cDNA clones. The overlapping sequences were assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a final sequence identified as SEQ ID NO:X. In the sixth column, “Total NT Seq.” refers to the total number of nucleotides in the contig sequence identified as SEQ ID NO:X.” The deposited clone may contain all or most of these sequences, reflected by the nucleotide position indicated as “5′ NT of Clone Seq.” (seventh column) and the “3′ NT of Clone Seq.” (eighth column) of SEQ ID NO:X. In the ninth column, the nucleotide position of SEQ ID NO:X of the putative start codon (methionine) is identified as “5′ NT of Start Codon.” Similarly, in column ten, the nucleotide position of SEQ ID NO:X of the predicted signal sequence is identified as “5′ NT of First AA of Signal Pep.” In the eleventh column, the translated amino acid sequence, beginning with the methionine, is identified as “AA SEQ ID NO:Y,” although other reading frames can also be routinely translated using known molecular biology techniques. The polypeptides produced by these alternative open reading frames are specifically contemplated by the present invention.

In the twelfth and thirteenth columns of Table 1A, the first and last amino acid position of SEQ ID NO:Y of the predicted signal peptide is identified as “First AA of Sig Pep” and “Last AA of Sig Pep.” In the fourteenth column, the predicted first amino acid position of SEQ ID NO:Y of the secreted portion is identified as “Predicted First AA of Secreted Portion”. The amino acid position of SEQ ID NO:Y of the last amino acid encoded by the open reading frame is identified in the fifteenth column as “Last AA of ORF”.

SEQ ID NO:X (where X may be any of the polynucleotide sequences disclosed in the sequence listing) and the translated SEQ ID NO:Y (where Y may be any of the polypeptide sequences disclosed in the sequence listing) are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO:X is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in the deposited clone. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used, for example, to generate antibodies which bind specifically to proteins containing the polypeptides and the secreted proteins encoded by the cDNA clones identified in Table 1A and/or elsewhere herein.

Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).

Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and the predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing a human cDNA of the invention deposited with the ATCC™, as set forth in Table 1A. The nucleotide sequence of each deposited plasmid can readily be determined by sequencing the deposited plasmid in accordance with known methods

The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular plasmid can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.

Also provided in Table 1A is the name of the vector which contains the cDNA plasmid. Each vector is routinely used in the art. The following additional information is provided for convenience.

Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBLUESCRIPT™ (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from STRATAGENE™ Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from STRATAGENE™.

Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from LIFE TECHNOLOGIES™, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from LIFE TECHNOLOGIES™. See, for instance, Gruber, C. E., et al., Focus 15:59 (1993). Vector lafmid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from LIFE TECHNOLOGIES™. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).

The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or a deposited cDNA (cDNA Clone ID). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include, but are not limited to, preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X and SEQ ID NO:Y using information from the sequences disclosed herein or the clones deposited with the ATCC™. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X and/or a cDNA contained in ATCC™ Deposit No:Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, and/or a polypeptide encoded by a cDNA contained in ATCC™ Deposit No:Z. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X and/or a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z, are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the complement of the coding strand of the cDNA contained in ATCC™ Deposit No:Z.

TABLE 1A 5′ NT of First Last ATCC ™ NT 5′ NT 3′ NT 5′ NT First AA AA AA First Last Deposit SEQ Total of of of AA of SEQ of of AA of AA Gene cDNA No: Z and ID NT Clone Clone Start Signal ID Sig Sig Secreted of No. Clone ID Date Vector NO: X Seq. Seq. Seq. Codon Pep NO: Y Pep Pep Portion ORF 1 HNFIP24 97903 pBluescript ™ 1 902 46 816 19 19 2 1 26 27 234 Feb. 26, 1997 209049 May 15, 1997 2 HETBY74 97904 Uni-ZAP XR 7 1923 30 1923 45 45 8 1 33 34 193 Feb. 26, 1997 209050 May 15, 1997 3 HTEEB42 97922 Uni-ZAP XR 9 1022 20 1022 59 59 10 1 22 23 298 Mar. 07, 1997 209070 May 22, 1997 4 HEMCM42 97978 Uni-ZAP XR 11 1041 48 1007 58 58 12 1 29 30 113 Mar. 27, 1997 209075 May 22, 1997 4 HEQCC55 209965 pCMVSport 13 1052 30 1052 62 62 14 1 27 28 112 Jun. 11, 1998 3.0 4 HEQCC55 209965 pCMVSport 15 1000 1 1000 25 25 16 1 27 28 129 Jun. 11, 1998 3.0 4 HEMCM42 97978 Uni-ZAP XR 15 1000 1 1000 25 25 16 1 27 28 129 Mar. 27, 1997 209075 May 22, 1997 4 HEQCC55 209965 pCMVSport 17 1037 1 1037 57 57 18 1 27 28 155 Jun. 11, 1998 3.0 5 HEMAE80 97975 Uni-ZAP XR 20 996 1 945 12 12 21 1 24 25 136 Apr. 04, 1997 209081 May 29, 1997 5 HEMAE80 97975 Uni-ZAP XR 22 1092 1 1092 61 61 23 1 24 25 136 Apr. 04, 1997 209081 May 29, 1997 6 HRDFB85 97977 Uni-ZAP XR 28 1705 23 1697 233 233 29 1 21 22 201 Apr. 04, 1997 209082 May 29, 1997 7 HDTAW95 209007 pCMVSport 38 1288 412 1288 571 571 39 1 16 Apr. 28, 1997 2.0 209083 May 29, 1997 8 HEMCV19 209010 Uni-ZAP XR 46 941 33 931 79 79 47 1 23 24 178 Apr. 28, 1997 209085 May 29, 1997 8 HEMCV19 209010 Uni-ZAP XR 48 736 1 736 84 84 49 1 22 23 181 Apr. 28, 1997 209085 May 29, 1997 8 HEMCV19 209010 Uni-ZAP XR 50 864 1 864 125 125 51 1 21 22 185 Apr. 28, 1997 209085 May 29, 1997 9 HETBX14 209010 Uni-ZAP XR 61 1292 303 1292 207 207 62 1 18 19 250 Apr. 28, 1997 209085 May 29, 1997 9 HETBX14 209010 Uni-ZAP XR 63 1146 157 1146 74 64 1 13 14 53 Apr. 28, 1997 209085 May 29, 1997 9 HETBX14 209010 Uni-ZAP XR 65 1146 157 1146 74 66 1 14 15 53 Apr. 28, 1997 209085 May 29, 1997 10 HLHSK94 209011 pBluescript ™ 70 1974 1 1794 112 112 71 1 26 27 379 Apr. 28, 1997 10 HLHSK94 209011 pBluescript 72 1789 1 1789 112 112 73 1 25 26 379 Apr. 28, 1997 10 HLHSK94 209011 pBluescript 74 1974 1 1794 112 112 75 1 25 26 379 Apr. 28, 1997 11 HLHFP03 209126 Uni-ZAP XR 76 613 1 613 224 224 77 1 20 21 116 Jun. 19, 1997 11 HLHFP03 209126 Uni-ZAP XR 78 613 1 613 224 224 79 1 19 20 116 Jun. 19, 1997 12 HHTLF25 209125 ZAP Express 83 697 1 661 142 142 84 1 26 27 111 Jun. 19, 1997 13 HTADX17 209124 Uni-ZAP XR 90 1140 22 1140 84 84 91 1 24 25 142 Jun. 19, 1997 13 HTADX17 209124 Uni-ZAP XR 92 1147 0 1148 92 92 93 1 23 24 142 Jun. 19, 1997 13 HTADX17 209124 Uni-ZAP XR 94 1140 22 1140 84 84 95 1 19 20 142 Jun. 19, 1997 14 HJACG02 209215 pBluescript 102 553 1 553 47 47 103 1 23 24 108 Aug. 21, 1997 SK− 14 HJACG02 209215 pBluescript 104 575 1 575 66 66 105 1 22 23 108 Aug. 21, 1997 SK− 15 HKGAJ54 209224 pSport1 107 1346 1 1346 31 31 108 1 27 28 303 Aug. 28, 1997 15 HKGAJ54 209224 pSport1 109 1332 1 1332 24 24 110 1 27 28 340 Aug. 28, 1997 16 HSVAK93 209563 Uni-ZAP XR 116 571 1 571 21 21 117 1 24 25 183 Dec. 18, 1997 16 HE8CH92 209580 Uni-ZAP XR 118 1282 1 1282 31 31 119 1 24 25 378 Jan. 14, 1998 16 HSVAK93 209563 Uni-ZAP XR 120 1240 1 1240 24 24 121 1 24 25 378 Dec. 18, 1997 17 HSDEK49 209603 Uni-ZAP XR 123 1590 96 1590 126 126 124 1 21 22 305 Jan. 29, 1998 17 HSDEK49 209603 Uni-ZAP XR 125 1782 1 1782 60 60 126 1 19 20 399 Jan. 29, 1998 18 HWBAO62 209603 pCMVSport 127 1903 1 1903 52 52 128 1 30 31 212 Jan. 29, 1998 3.0 18 HWBAO62 209603 pCMVSport 129 1940 1 1940 81 81 130 1 30 31 101 Jan. 29, 1998 3.0 19 HWHGU54 209782 pCMVSport 131 1445 1 1445 145 145 132 1 19 20 414 Apr. 20, 1998 3.0 20 HCEJQ69 209782 Uni-ZAP XR 134 1777 1 1777 39 39 135 1 26 27 473 Apr. 20, 1998 20 HCEJQ69 209782 Uni-ZAP XR 136 1774 1 1774 39 39 137 1 26 27 550 Apr. 20, 1998 20 HCEJQ69 209782 Uni-ZAP XR 138 1777 1 1777 39 39 139 1 26 27 380 Apr. 20, 1998 21 HT5GJ57 209889, UNI-ZAP ™ 159 1797 92 1797 122 122 160 1 25 26 190 May 22, 1998 XR 21 HT5GJ57 209889 Uni-ZAP XR 161 1773 1 1773 105 105 162 1 25 26 243 May 22, 1998 22 HPIBX03 209965 Uni-ZAP XR 169 2209 1 2178 81 81 170 1 29 30 709 Jun. 11, 1998 23 HDPBO81 203070 pCMVSport 174 3798 1 3798 265 265 175 1 26 27 348 Jul. 27, 1998 3.0 23 HDPBO81 203070 pCMVSport 176 3793 1 3793 255 255 177 1 26 27 348 Jul. 27, 1998 3.0 24 HWBFY57 203648 pCMVSport 179 1796 1 1796 113 113 180 1 19 20 290 Feb. 09, 1999 3.0 25 HYABV21 PTA-3105 pCMVSport 182 2738 1 2738 55 55 183 1 16 17 301 Feb. 23, 2001 3.0 25 HYABV21 PTA-3105 pCMVSport 184 729 28 729 63 63 185 1 16 17 222 Feb. 23, 2001 3.0 26 HOHBY69 203331 pCMVSport 186 4995 1 4995 82 82 187 1 22 23 1189 Oct. 08, 1998 2.0 26 HOHBY69 203331 pCMVSport 188 4631 1 4631 84 84 189 1 22 23 1034 Oct. 08, 1998 2.0 27 HDHMA45 203331 pCMVSport 202 2184 1 2184 199 199 203 1 33 34 413 Oct. 08, 1998 2.0 27 HDHMA45 203331 pCMVSport 204 2190 1 2190 204 204 205 1 33 34 413 Oct. 08, 1998 2.0 28 HMADJ14 PTA-622 Uni-ZAP XR 206 1364 15 1364 278 278 207 1 68 69 352 Sep. 02, 1999 28 HMADJ14 PTA-622 Uni-ZAP XR 208 1583 1 1583 264 264 209 1 25 26 257 Sep. 02, 1999 28 HMADJ14 PTA-622 Uni-ZAP XR 210 1444 91 1444 125 125 211 1 25 26 257 Sep. 02, 1999 28 HMADJ14 203979 Uni-ZAP XR 212 1444 91 1444 125 125 213 1 25 26 257 Apr. 29, 1999 28 HMADJ14 PTA-622 Uni-ZAP XR 214 1892 1 1891 264 264 215 1 25 26 291 Sep. 02, 1999 28 HMADJ14 PTA-622 Uni-ZAP XR 216 1439 1 1439 47 47 217 1 1 2 242 Sep. 02, 1999 29 HEAAL31 97903 Uni-ZAP XR 218 991 374 970 60 60 219 1 24 25 177 Feb. 26, 1997 209049 May 15, 1997 29 HEAAL31 97903 Uni-ZAP XR 220 2416 1387 2413 1473 1473 221 1 18 19 25 Feb. 26, 1997 209049 May 15, 1997 30 HEMFA84 203499 Uni-ZAP XR 222 985 1 985 42 42 223 1 17 18 257 Dec. 01, 1998 31 HDPPA04 PTA-867 pCMVSport 224 2406 1 2406 271 271 225 1 19 20 283 Oct. 26, 1999 3.0 31 HDPPA04 PTA-867 pCMVSport 226 1675 1 1613 1003 227 1 11 12 23 Oct. 26, 1999 3.0 31 HDPPA04 PTA-867 pCMVSport 228 786 1 786 261 261 229 1 19 20 93 Oct. 26, 1999 3.0 32 HE2OA95 203960 Uni-ZAP XR 230 1671 1 1671 1224 231 1 9 10 35 Apr. 26, 1999 33 HKABZ65 209683 pCMVSport 232 1189 1 1189 77 77 233 1 17 18 243 Mar. 20, 1998 2.0 33 HKABZ65 209683 pCMVSport 234 1191 1 1191 69 69 235 1 17 18 243 Mar. 20, 1998 2.0

Tables 1B.1 and 1B.2

The first column in Tables 1B.1 and 1B.2 provides the gene number in the application corresponding to the clone identifier. The second column in Tables 1B.1 and 1B.2 provides a unique “Clone ID:” for the cDNA clone related to each contig sequence disclosed in Tables 1B.1 and 1B.2. This clone ID references the cDNA clone which contains at least the 5′ most sequence of the assembled contig and at least a portion of SEQ ID NO:X was determined by directly sequencing the referenced clone. The referenced clone may have more sequence than described in the sequence listing or the clone may have less. In the vast majority of cases, however, the clone is believed to encode a full-length polypeptide. In the case where a clone is not full-length, a full-length cDNA can be obtained by methods described elsewhere herein. The third column in Tables 1B.1 and 1B.2 provides a unique “Contig ID” identification for each contig sequence. The fourth column in Tables 1B.1 and 1B.2 provides the “SEQ ID NO:” identifier for each of the contig polynucleotide sequences disclosed in Tables 1B.1 and 1B.2. Table 1B.1

The fifth column in Table 1B.1, “ORF (From-To)”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence “SEQ ID NO:X” that delineate the preferred open reading frame (ORF) shown in the sequence listing and referenced in Table 1B.1, column 6, as SEQ ID NO:Y. Where the nucleotide position number “To” is lower than the nucleotide position number “From”, the preferred ORF is the reverse complement of the referenced polynucleotide sequence. The sixth column in Table 1B.1 provides the corresponding SEQ ID NO:Y for the polypeptide sequence encoded by the preferred ORF delineated in column 5. In one embodiment, the invention provides an amino acid sequence comprising, or alternatively consisting of, a polypeptide encoded by the portion of SEQ ID NO:X delineated by “ORF (From-To)”. Also provided are polynucleotides encoding such amino acid sequences and the complementary strand thereto. Column 7 in Table 1B.1 lists residues comprising epitopes contained in the polypeptides encoded by the preferred ORF (SEQ ID NO:Y), as predicted using the algorithm of Jameson and Wolf, (1988) Comp. Appl. Biosci. 4:181-186. The Jameson-Wolf antigenic analysis was performed using the computer program PROTEAN (Version 3.11 for the Power MacIntosh, DNASTAR, Inc., 1228 South Park Street Madison, Wis.). In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, at least one, two, three, four, five or more of the predicted epitopes as described in Table 1B.1. It will be appreciated that depending on the analytical criteria used to predict antigenic determinants, the exact address of the determinant may vary slightly.

Column 8 in Table 1B.1 (“Cytologic Band”) provides a chromosomal map location for certain polynucleotides of the invention (e.g., polynucleotides corresponding to SEQ ID NO:X). Chromosomal location was determined by finding exact matches to EST and cDNA sequences contained in the NCBI (National Center for Biotechnology Information) UniGene database. Each sequence in the UniGene database is assigned to a “cluster”; all of the ESTs, cDNAs, and STSs in a cluster are believed to be derived from a single gene. Chromosomal mapping data is often available for one or more sequence(s) in a UniGene cluster; this data (if consistent) is then applied to the cluster as a whole. Thus, it is possible to infer the chromosomal location of a new polynucleotide sequence by determining its identity with a mapped UniGene cluster.

A modified version of the computer program BLASTN (Altshul, et al., J. Mol. Biol. 215:403-410 (1990), and Gish, and States, Nat. Genet. 3:266-272) (1993) was used to search the UniGene database for EST or cDNA sequences that contain exact or near-exact matches to a polynucleotide sequence of the invention (the ‘Query’). A sequence from the UniGene database (the ‘Subject’) was said to be an exact match if it contained a segment of 50 nucleotides in length such that 48 of those nucleotides were in the same order as found in the Query sequence. If all of the matches that met this criteria were in the same UniGene cluster, and mapping data was available for this cluster, it is indicated in Table 1B.1 under the heading “Cytologic Band”. Where a cluster had been further localized to a distinct cytologic band, that band is disclosed; where no banding information was available, but the gene had been localized to a single chromosome, the chromosome is disclosed.

Once a presumptive chromosomal location was determined for a polynucleotide of the invention, an associated disease locus was identified by comparison with a database of diseases which have been experimentally associated with genetic loci. The database used was the Morbid Map, derived from OMIM™ (“Online Mendelian Inheritance in Man”; McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.) 2000; world wide web at ncbi.nlm.nih.gov/omim/). If the putative chromosomal location of a polynucleotide of the invention (Query sequence) was associated with a disease in the Morbid Map database, an OMIM reference identification number was noted in column 9, Table 1B.1, labeled “OMIM Disease Reference(s). Table 5 is a key to the OMIM reference identification numbers (Table 5, column 1), and provides a description of the associated disease in Table 5, column 2. Table 1B.2

Column 5, in Table 1B.2, provides an expression profile and library code:count for each of the contig sequences (SEQ ID NO:X) disclosed in Table 1B.2, which can routinely be combined with the information provided in Table 4 and used to determine the tissues, cells, and/or cell line libraries which predominantly express the polynucleotides of the invention. The first number in Table 1B.2, column 5 (preceding the colon), represents the tissue/cell source identifier code corresponding to the code and description provided in Table 4. The second number in column 5 (following the colon) represents the number of times a sequence corresponding to the reference polynucleotide sequence was identified in the corresponding tissue/cell source. Those tissue/cell source identifier codes in which the first two letters are “AR” designate information generated using DNA array technology. Utilizing this technology, cDNAs were amplified by PCR and then transferred, in duplicate, onto the array. Gene expression was assayed through hybridization of first strand cDNA probes to the DNA array. cDNA probes were generated from total RNA extracted from a variety of different tissues and cell lines. Probe synthesis was performed in the presence of ³³P dCTP, using oligo (dT) to prime reverse transcription. After hybridization, high stringency washing conditions were employed to remove non-specific hybrids from the array. The remaining signal, emanating from each gene target, was measured using a Phosphorimager. Gene expression was reported as Phosphor Stimulating Luminescence (PSL) which reflects the level of phosphor signal generated from the probe hybridized to each of the gene targets represented on the array. A local background signal subtraction was performed before the total signal generated from each array was used to normalize gene expression between the different hybridizations. The value presented after “[array code]:” represents the mean of the duplicate values, following background subtraction and probe normalization. One of skill in the art could routinely use this information to identify normal and/or diseased tissue(s) which show a predominant expression pattern of the corresponding polynucleotide of the invention or to identify polynucleotides which show predominant and/or specific tissue and/or cell expression.

TABLE 1B.1 ORF AA Gene cDNA Contig SEQ ID (From- SEQ ID Predicted Cytologic No: Clone ID ID: NO: X To) NO: Y Epitopes Band OMIM Disease Reference(s) 3 HTEEB42 206980 9 59-952 10 Met-1 to His-7. 21q21.2 4 HEMCM42 407085 11 58-399 12 Pro-35 to Trp- 42, Ala-53 to Asp-62, Arg- 103 to Pro-113. 4 HEMCM42 1352150 15 25-411 16 Pro-35 to Trp- 42, Ala-53 to Asp-62, Arg- 103 to Phe-110, Ile-114 to Glu- 120. 4 HEQCC55 884824 13 62-397 14 Pro-35 to Trp- 42, Pro-65 to Asp-72, Thr-86 to Phe-93, Ile- 97 to Glu-103. 4 HEQCC55 1352368 15 25-411 16 Pro-35 to Trp- 16p13.3 141750, 141800, 141800, 42, Ala-53 to 141800, 141800, 141850, Asp-62, Arg- 141850, 141850, 141850, 103 to Phe-110, 141850, 156850, 186580, Ile-114 to Glu- 191092, 600140, 600273, 120. 601313, 601785 4 HEQCC55 748227 17 57-524 18 Pro-35 to Trp- 42, Pro-65 to Asp-72, Thr-86 to Glu-92, Pro- 96 to Gly-104, Ser-138 to Gly- 154. 5 HEMAE80 409495 20 12-422 21 Ser-91 to Lys- 98. 5 HEMAE80 1310948 22 61-471 23 Ser-91 to Lys- 4p16-p15 225500, 600593, 602363 98. 6 HRDFB85 411020 28 233-838  29 Gly-147 to Met- 7q11.23 116860, 129900, 233700, 152, Cys-177 to 600079 Lys-188. 7 HDTAW95 412472 38 571-621  39 8 HEMCV19 423219 46 79-615 47 Thr-19 to Ala- 33, Leu-54 to Asp-82, Pro-89 to Ala-97, Pro- 100 to Lys-125, Ser-127 to Phe- 135, Gly-164 to Leu-169, Cys- 173 to Arg-178. 8 HEMCV19 1352162 48 84-626 49 Thr-19 to Ser- 19q12-q13.1 164731, 172400, 172400, 31, Leu-54 to 180901, 180901, 221770, Asp-82, Pro-89 248600, 600918, 602716 to Ala-97, Pro- 100 to Lys-125, Ser-127 to Phe- 135. 8 HEMCV19 1352161 50 125-679  51 Thr-19 to Ala- 33, Leu-54 to Asp-82, Pro-89 to Ala-97, Pro- 100 to Lys-125, Ser-127 to Phe- 135, Ser-180 to Ser-185. 9 HETBX14 806447 61 207-956  62 Glu-27 to Trp- 19q13.3-q13.4 113900, 126340, 126391, 35, Leu-77 to 130410, 134790, 138570, Ala-89, Pro-96 160900, 173850, 191044, to Asn-109, 258501, 600040, 600138, Ser-149 to Arg- 602225, 602225 156, Gln-172 to Ile-182, Glu- 193 to Gly-204, Glu-245 to Asn- 250. 9 HETBX14 422659 63 74-235 64 Gly-12 to Ser- 22, Pro-34 to Ser-53. 10 HLHSK94 1307727 72 112-1251 73 Gly-29 to Glu- 12q14.3 181430, 600808, 602116 34, Arg-71 to Arg-76, Thr- 176 to Cys-182, Gly-184 to Glu- 199, Lys-277 to Lys-287, Ser- 292 to Cys-305, Gly-318 to Tyr- 341, Gln-358 to Tyr-377. 10 HLHSK94 422828 74 112-1251 75 Gly-29 to Glu- 34, Arg-71 to Arg-76, Thr- 176 to Cys-182, Gly-184 to Glu- 199. 11 HLHFP03 460467 76 224-574  77 Tyr-28 to Phe- 34, Thr-54 to Val-60, Tyr-73 to Thr-82. 11 HLHFP03 460467 78 224-574  79 Tyr-28 to Phe- 34, Thr-54 to Val-60, Tyr-73 to Thr-82. 12 HHTLF25 461438 83 142-474  84 Ala-28 to Ser- 19q13.1 164731, 172400, 172400, 33, Ala-76 to 180901, 180901, 221770, Lys-111. 248600, 600918, 602716 13 HTADX17 457172 90 84-512 91 Glu-15 to Arg- 23, Asn-79 to Gly-84. 13 HTADX17 753289 92 92-520 93 Glu-15 to Arg- 1q23.1 107300, 131210, 136132, 23, Asn-79 to 145001, 173610, 601652 Gly-84, Ser-101 to Gly-106, Ser- 111 to Asn-116. 13 HTADX17 457172 94 84-512 95 Glu-15 to Arg- 23, Asn-79 to Gly-84. 14 HJACG02 509948 102 47-373 103 Val-54 to Asp- 59. 14 HJACG02 1307789 104 66-392 105 Val-54 to Asp- 19p13.3 108725, 120700, 133171, 59. 136836, 145981, 147141, 164953, 188070, 600957, 601238, 601846, 602216, 602477 15 HKGAJ54 498303 107 31-942 108 Ala-55 to Thr- 62, His-164 to Gly-175, Ala- 197 to Glu-202. 15 HKGAJ54 1300770 109  24-1046 110 Ala-55 to Thr- 62, His-164 to Gly-175, Ala- 197 to Thr-204, Ser-212 to Ala- 220, Gln-226 to Glu-233, Pro- 252 to Gly-263, Arg-318 to Glu- 326, Ser-331 to Pro-340. 16 HE8CH92 609866 118  31-1167 119 Ser-44 to Ser-51, Cys-53 to Cys- 64, Val-76 to Lys-83, Pro-102 to Gly-108, Arg-133 to Thr- 162, Thr-204 to Ala-209, Asp- 235 to Glu-241, Lys-270 to Ala- 282, Ala-286 to Gly-297, Ser- 346 to Arg-351, Gly-368 to Gly- 374. 16 HSVAK93 597462 116 21-569 117 Ser-44 to Ser-51, Cys-53 to Cys- 64, Val-76 to Lys-83, Pro-102 to Gly-108, Arg-133 to Thr- 162, Thr-169 to Lys-183. 16 HSVAK93 1352228 120  24-1157 121 Ser-44 to Ser-51, Cys-53 to Cys- 64, Val-76 to Lys-83, Pro-102 to Gly-108, Arg-133 to Thr- 162, Thr-204 to Ala-209, Asp- 235 to Glu-241, Lys-270 to Ala- 282, Ala-286 to Gly-297, Ser- 346 to Arg-351, Gly-368 to Gly- 374. 17 HSDEK49 625998 123 126-1043 124 Val-29 to Val- 37, Asp-71 to His-76, Gln-78 to Gly-84, Met- 105 to His-110, Trp-117 to Gly- 122, Gln-136 to Lys-141, Leu- 143 to Ala-149, Thr-162 to Asp- 174, Ser-181 to Lys-186, Arg- 214 to Glu-220, Glu-232 to Glu- 238, Cys-249 to Asp-265. 17 HSDEK49 1352253 125  60-1256 126 Val-29 to Val- Xq12-q13.3 300011, 300011, 300011, 37, Asp-71 to 300127, 305450, 309605, His-76, Gln-78 313700, 313700, 313700, to Gly-84, Met- 313700, 313700, 314580 105 to His-110, Trp-117 to Asn- 123, Lys-179 to Pro-187, Gly- 218 to Asp-224, Leu-237 to Ala- 243, Thr-256 to Asp-268, Ser- 275 to Lys-280, Arg-308 to Glu- 314, Glu-326 to Glu-332, Cys- 343 to Asp-359. 18 HWBAO62 838164 127 52-687 128 Ile-40 to Glu-45, Cys-63 to Val- 69, Glu-83 to Asn-94, Pro- 107 to Cys-115, Phe-137 to Ser- 143, Ser-159 to Thr-167, Glu- 200 to Tyr-210. 18 HWBAO62 625914 129 81-386 130 Ile-40 to Glu-45, Cys-63 to Val- 69, Glu-83 to Phe-95. 19 HWHGU54 695695 131 145-1389 132 Phe-25 to Tyr- 30, Gln-37 to Arg-42, Lys- 106 to Leu-112, Leu-123 to Leu- 130, Gln-142 to Phe-150, Gln- 183 to Lys-188, Asp-219 to Glu- 226, Lys-359 to Glu-366. 20 HCEJQ69 1243825 134  39-1457 135 Thr-41 to Gly- 104170, 104170, 104170, 47, Pro-170 to 115470, 142360, 188400, Asp-176, Arg- 188400, 217095, 600850, 257 to Trp-262, 601607 Gln-276 to Ser- 283, Phe-278 to Val-285, Arg- 323 to Gly-331, Glu-348 to Ser- 354, Arg-362 to Gly-385, Pro- 364 to Arg-377, Arg-407 to Leu- 431, Pro-409 to Ser-427, Gly- 438 to Gly-448, Gly-440 to Ala- 449. 20 HCEJQ69 1243825 134  39-1457 135 Thr-41 to Gly- 22q11 104170, 104170, 104170, 47, Pro-170 to 115470, 142360, 188400, Asp-176, Leu- 188400, 217095, 600850, 257 to Trp-262, 601607 Gln-276 to Ser- 283, Arg-323 to Leu-330, Pro- 364 to Arg-377, Arg-407 to Leu- 431, Gly-438 to Gly-448. 20 HCEJQ69 872582 136  39-1688 137 Thr-41 to Gly- 47, Pro-170 to Asp-176, Arg- 257 to Trp-262, Gln-276 to Ser- 283, Phe-278 to Val-285, Arg- 323 to Gly-331, Glu-348 to Ser- 354, Arg-362 to Gly-385, Pro- 364 to Arg-377, Arg-407 to Leu- 431, Pro-409 to Ser-427, Gly- 438 to Gly-448, Gly-440 to Ala- 449, Pro-500 to Ser-514, Pro- 521 to His-528. 20 HCEJQ69 872582 136  39-1688 137 Thr-41 to Gly- 47, Pro-170 to Asp-176, Leu- 257 to Trp-262, Gln-276 to Ser- 283, Arg-323 to Leu-330, Pro- 364 to Phe-374, Pro-401 to Ser- 409, Arg-415 to Arg-427, Ala- 474 to Gln-484, Pro-500 to Ser- 514, Pro-521 to His-528. 20 HCEJQ69 609999 138  39-1178 139 Thr-41 to Gly- 47, Pro-170 to Asp-176, Arg- 257 to Trp-262, Gln-276 to Ser- 283, Phe-278 to Val-285, Arg- 323 to Gly-331, Glu-348 to Ser- 354. 20 HCEJQ69 609999 138  39-1178 139 Thr-41 to Gly- 47, Pro-170 to Asp-176, Leu- 257 to Trp-262, Gln-276 to Ser- 283, Arg-323 to Leu-330, Pro- 362 to Val-374. 21 HT5GJ57 740767 159 122-694  160 Ser-29 to Thr- 7q11.23 116860, 129900, 233700, 57, Pro-74 to 600079 Lys-79, Pro-85 to Glu-107, Tyr- 118 to Tyr-136, Gln-144 to Gln- 152, Ala-182 to Glu-188. 21 HT5GJ57 1299921 161 105-836  162 Ser-29 to Thr- 7q11.23 116860, 129900, 233700, 57, Pro-74 to 600079 Lys-79, Pro-85 to Glu-107, Tyr-118 to Tyr- 136, Gln-144 to Gln-152, Ala- 182 to Asn-195, Arg-203 to Val- 208, Leu-212 to Ser-217, Gly- 222 to Val-234. 22 HPIBX03 743314 169  81-2207 170 3p24.3-p22.1 154705, 182280, 190160, 227646, 261510, 600163, 601154 23 HDPBO81 892018 174 265-1308 175 Asp-53 to Tyr- 61, Pro-105 to Ile-128, Arg- 133 to Leu-140, Gln-182 to Ala- 188, Pro-205 to Asn-218, Gly- 259 to Ala-264, Asn-290 to Ser- 302, Glu-307 to Tyr-314, Tyr- 317 to Lys-332. 23 HDPBO81 790188 176 255-1301 177 Asp-53 to Tyr- 61, Pro-105 to Ile-128, Arg- 133 to Leu-140, Gln-182 to Ala- 188, Pro-205 to Asn-218, Gly- 259 to Ser-264. 24 HWBFY57 837478 179 113-985  180 Ser-69 to Arg- 79, Ile-82 to Arg-89, Pro-129 to Ser-137, Leu- 146 to Lys-151. 25 HYABV21 1281466 182 55-960 183 25 HYABV21 1213593 184 63-728 185 26 HOHBY69 827480 186  82-3648 187 Phe-23 to Arg- 15q22.3-q23 118485, 151670, 231680, 31, Leu-62 to 272800, 272800, 272800, Asp-72, Val-96 276700, 600374, 601780 to Asp-101, Thr-111 to Asn- 116, Glu-128 to Thr-135, Val- 142 to Ser-149, Asn-217 to Val- 222, Glu-233 to Arg-241, Gly- 272 to Leu-280, Gln-286 to Thr- 293, Tyr-303 to Ile-308, Gly- 354 to Thr-360, Glu-408 to Lys- 419, Glu-508 to Lys-514, Arg- 521 to Val-526, Gly-529 to Phe- 542, Asp-551 to Tyr-557, Thr- 587 to Thr-593, His-656 to Asp- 665, Met-697 to Arg-705, Asp- 709 to Thr-716, Glu-755 to Gly- 760, Asn-779 to His-786, Leu- 810 to Asp-816, Leu-844 to Ala- 851, Gln-871 to Gly-877, Glu- 884 to Gln-889, Ser-931 to Asn- 943, Ser-974 to Ile-982, Gly- 1039 to Gln- 1058, Arg-1121 to Arg-1127, Ser-1134 to Trp- 1139, Ser-1172 to Pro-1183. 26 HOHBY69 815681 188  84-3185 189 Phe-23 to Arg- 31, Leu-62 to Asp-72, Val-96 to Asp-101, Thr-111 to Asn- 116, Glu-128 to Thr-135, Val- 142 to Ser-149, Asn-217 to Val- 222, Glu-233 to Arg-241, Gly- 272 to Leu-280, Gln-286 to Thr- 293, Tyr-303 to Ile-308, Gly- 354 to Thr-360, Glu-408 to Lys- 419, Glu-508 to Lys-514, Arg- 521 to Val-526, Gly-529 to Phe- 542, Asp-551 to Tyr-557, Thr- 587 to Thr-593, His-656 to Asp- 665, Met-697 to Arg-705, Asp- 709 to Thr-716, Glu-755 to Gly- 760, Asn-779 to His-786, Leu- 810 to Asp-816, Leu-844 to Ala- 851, Gln-871 to Gly-877, Glu- 884 to Gln-889, Ser-931 to Asn- 943, Ser-974 to Ile-982. 27 HDHMA45 902513 202 199-1440 203 Ala-145 to Ser- 11q 154, Ala-258 to Tyr-263, Ala- 287 to Arg-297, Thr-306 to Met- 316. 27 HDHMA45 812764 204 204-1445 205 Ala-145 to Ser- 154, Ala-258 to Tyr-263, Ala- 287 to Arg-297, Thr-306 to Met- 316. 28 HMADJ14 1099342 206 278-1333 207 Asp-229 to Gln- 8q22 216550, 259730 236, Asn-244 to Lys-250, Trp- 258 to Asn-266. 28 HMADJ14 889659 208 264-1037 209 Asp-229 to Gln- 236, Asn-244 to Phe-251. 28 HMADJ14 843725 212 125-898  213 Asp-229 to Gln- 8q22 216550, 259730 236, Asn-244 to Phe-251. 28 HMADJ14 795479 214 264-1139 215 Asp-229 to Cys- 239, Asn-244 to Glu-253, Thr- 271 to Ser-278. 28 HMADJ14 426068 216 47-772 217 30 HEMFA84 608198 222 42-815 223 Leu-23 to Asp- 17p13.3 113721, 247200, 600059, 34, Cys-97 to 601545 Pro-106, Ser- 202 to Gly-208, Pro-251 to Gly- 257. 31 HDPPA04 904765 224 271-1122 225 Lys-61 to Arg- 72, Arg-95 to Tyr-100, Ala- 121 to Ile-126, Asn-163 to Gly- 172, Lys-183 to Asn-189, Ser- 211 to His-218, Leu-251 to Val- 269. 31 HDPPA04 905419 226 1003-1074  227 Ser-16 to Lys- 23. 31 HDPPA04 905418 228 261-542  229 Lys-61 to Arg- 72. 32 HE2OA95 637595 230 1224-1331  231 2q32.3 600258, 602087 33 HKABZ65 862030 232 77-808 233 Ser-25 to Ala- 31, Gln-146 to Ser-151, His- 231 to Asn-236. 33 HKABZ65 665424 234 69-800 235 Ser-25 to Ala- 31, Gln-146 to Ser-151, His- 231 to Asn-236.

TABLE 1B.2 cDNA Clone Tissue Distribution Library Code:Count (see Table 4 Gene No: ID Contig ID: SEQ ID NO: X for Library Codes) 3 HTEEB42 206980 9 AR174: 12, AR191: 12, AR190: 11, AR244: 11, AR181: 11, AR291: 10, AR186: 10, AR180: 10, AR175: 10, AR192: 10, AR1899, AR1769, AR2409, AR2699, AR2419, AR1789, AR2709, AR1778, AR2668, AR2688, AR1838, AR2738, AR2748, AR1658, AR2477, AR1647, AR1987, AR1847, AR1667, AR1627, AR2027, AR1617, AR1637, AR2467, AR2456, AR1976, AR2896, AR1736, AR2016, AR2676, AR1826, AR2716, AR0526, AR2066, AR3096, AR1856, AR1886, AR2756, AR2636, AR2515, AR2365, AR2845, AR1945, AR2955, AR2555, AR2355, AR2775, AR2995, AR1795, AR0555, AR2905, AR1045, AR0335, AR1935, AR2284, AR2304, AR2044, AR1964, AR1704, AR2854, AR2564, AR1724, AR2724, AR2574, AR2624, AR2054, AR2334, AR3084, AR2614, AR1954, AR2524, AR3004, AR2234, AR0894, AR2874, AR2384, AR2434, AR2144, AR2964, AR2374, AR2654, AR2504, AR2394, AR2884, AR2984, AR2243, AR2943, AR2293, AR2483, AR3163, AR2073, AR2863, AR3123, AR2973, AR2643, AR1993, AR0613, AR2933, AR0533, AR2273, AR0603, AR3113, AR2113, AR2493, AR2253, AR2923, AR2193, AR2583, AR0393, AR2153, AR3133, AR2823, AR2263, AR2603, AR2312, AR2422, AR2032, AR1712, AR1682, AR2102, AR2002, AR2592, AR2342, AR0962, AR2322, AR1692, AR2222, AR2542, AR2182, AR2212, AR2532, AR2832, AR2131, AR2161, AR2171, AR3101 L0794: 4, H0624: 2, H0038: 2, L0375: 2, S0330: 2, L0750: 2, L0779: 2, H0031: 1, H0644: 1, H0124: 1, H0591: 1, H0616: 1, H0264: 1, H0623: 1, L0770: 1, L0637: 1, L0805: 1, L0663: 1, L0749: 1, L0777: 1, L0780: 1 and L0599: 1. 4 HEMCM42 407085 11 4 HEQCC55 884824 13 4 HEQCC55 1352368 15 AR216: 11, AR217: 10, AR2149, AR2079, AR2638, AR1958, AR1658, AR2538, AR2248, AR2428, AR0538, AR1648, AR1638, AR2468, AR1618, AR2228, AR2458, AR1668, AR1708, AR1628, AR3087, AR1977, AR2127, AR3097, AR2237, AR3127, AR1987, AR3116, AR2506, AR2546, AR2056, AR2436, AR2136, AR2746, AR1686, AR2646, AR1935, AR2965, AR2015, AR2725, AR2385, AR0335, AR2755, AR1755, AR2824, AR3134, AR2214, AR2914, AR2834, AR2254, AR1744, AR2354, AR1044, AR2614, AR1714, AR2774, AR2974, AR2884, AR1774, AR3004, AR1834, AR2954, AR1694, AR3164, AR1924, AR2704, AR1814, AR0894, AR2664, AR2893, AR2693, AR1783, AR2263, AR1733, AR1723, AR2393, AR2683, AR2993, AR2903, AR2933, AR1893, AR1963, AR1853, AR2313, AR2573, AR2403, AR2853, AR2473, AR1763, AR0393, AR2103, AR2713, AR2553, AR1913, AR2673, AR2043, AR1823, AR0963, AR2623, AR2003, AR1793, AR2373, AR2273, AR1993, AR2863, AR0603, AR2343, AR2333, AR2323, AR0612, AR1902, AR2942, AR2872, AR2582, AR1882, AR2292, AR0552, AR2302, AR2152, AR2282, AR2032, AR2362, AR2112, AR2192, AR2181, AR2561 L0803: 5, L0755: 5, L0666: 4, S0418: 3, H0059: 3, H0494: 3, S0420: 2, H0086: 2, H0551: 2, H0413: 2, L0763: 2, L3904: 2, L0646: 2, L0800: 2, L0775: 2, L0659: 2, L0809: 2, H0144: 2, H0435: 2, H0670: 2, L0731: 2, S0342: 1, H0294: 1, S0180: 1, H0734: 1, S0046: 1, S0278: 1, H0437: 1, H0392: 1, H0544: 1, H0545: 1, L0471: 1, H0012: 1, H0375: 1, H0286: 1, S0250: 1, H0039: 1, H0553: 1, H0628: 1, H0646: 1, L0769: 1, L5565: 1, L0761: 1, L0764: 1, L0773: 1, L0662: 1, L0649: 1, L0804: 1, L0774: 1, L0806: 1, L0653: 1, L0657: 1, L0512: 1, L0789: 1, L0663: 1, S0406: 1, L0743: 1, L0754: 1, L0750: 1, L0780: 1, L0581: 1, L0603: 1 and H0665: 1. 4 HEMCM42 1352150 15 AR205: 54, AR202: 41, AR206: 35, AR204: 29, AR244: 19, AR192: 15, AR194: 11, AR296: 10, AR198: 10, AR274: 10, AR2319, AR2419, AR2468, AR1867, AR0606, AR1046, AR1826, AR2346, AR2896, AR2515, AR2435, AR0525, AR2824, AR0614, AR2194, AR2664, AR0534, AR0554, AR2704, AR2923, AR2683, AR0393, AR2983, AR2933, AR2903, AR2693, AR3163, AR2833, AR1833, AR0333, AR2372, AR0892, AR2482, AR1772, AR2292, AR2182, AR2402, AR3102, AR3122, AR3002, AR1752, AR2772, AR2672, AR3131, AR1851, AR2561, AR2991, AR0961 H0494: 9, L0803: 5, L0755: 5, S0418: 4, H0551: 4, L3904: 4, L0666: 4, L2260: 4, H0435: 4, H0734: 3, H0510: 3, H0059: 3, S0436: 3, S0420: 2, H0545: 2, H0086: 2, H0628: 2, H0413: 2, L0763: 2, L0646: 2, L0800: 2, L0775: 2, L0659: 2, L0809: 2, L0663: 2, H0144: 2, H0670: 2, S0380: 2, S0406: 2, L0731: 2, L0581: 2, S0342: 1, H0294: 1, S0180: 1, S0212: 1, H0484: 1, S0408: 1, S0046: 1, H0393: 1, S0278: 1, H0437: 1, H0392: 1, H0587: 1, H0599: 1, H0748: 1, H0544: 1, L0471: 1, H0012: 1, H0375: 1, H0286: 1, S0250: 1, H0039: 1, H0553: 1, H0646: 1, H0652: 1, S0144: 1, S0422: 1, L0769: 1, L5565: 1, L0761: 1, L0764: 1, L0773: 1, L0662: 1, L0649: 1, L0804: 1, L0774: 1, L0806: 1, L0653: 1, L0657: 1, L0512: 1, L0789: 1, H0519: 1, H0593: 1, S0126: 1, H0684: 1, H0672: 1, H0555: 1, L0743: 1, L0754: 1, L0750: 1, L0780: 1, L0603: 1 and H0665: 1. 4 HEQCC55 748227 17 5 HEMAE80 409495 20 5 HEMAE80 1310948 22 AR055: 113, AR060: 64, AR185: 56, AR283: 50, AR299: 49, AR300: 47, AR240: 43, AR277: 42, AR282: 42, AR104: 41, AR039: 41, AR089: 36, AR316: 33, AR218: 31, AR219: 31, AR096: 28, AR313: 16 S0126: 7, L0748: 6, S0338: 3, L0759: 3, S0318: 2, S0046: 1, H0251: 1, S0334: 1, S0336: 1, S0316: 1, S0312: 1, S0314: 1, H0056: 1, L0769: 1, L0659: 1, L0747: 1, L0749: 1, L0750: 1, L0777: 1, L0780: 1, S0194: 1 and S0276: 1. 6 HRDFB85 411020 28 AR205: 15, AR1949, AR2448, AR2467, AR2737, AR2637, AR2046, AR2705, AR2435, AR2065, AR3105, AR2745, AR1984, AR2414, AR1864, AR1824, AR0394, AR3094, AR3123, AR2823, AR2473, AR2533, AR2403, AR0893, AR2713, AR2893, AR0523, AR2693, AR2773, AR0603, AR2513, AR2653, AR1833, AR3163, AR1923, AR0532, AR2982, AR2672, AR2842, AR2992, AR0612, AR1842, AR1752, AR2132, AR2342, AR2682, AR0552, AR2952, AR2752, AR2662, AR2272, AR2492, AR2932, AR3002, AR0332, AR2912, AR2942, AR2902, AR1852, AR2312, AR2852, AR3132, AR0962, AR2962, AR2382, AR2261, AR2371, AR2331, AR2181, AR2291, AR2811, AR2861, AR1041, AR2831, AR2921, AR2591, AR2561 S0436: 29, S0406: 23, S0440: 20, S0442: 16, S0358: 15, H0622: 15, S0354: 14, S0408: 12, S0444: 11, H0435: 11, S0360: 10, S0356: 9, H0039: 9, L0666: 9, L0751: 9, H0553: 8, H0506: 8, H0689: 7, L0748: 7, H0661: 6, L0659: 6, L0809: 6, L0665: 6, S0376: 5, L3818: 5, L0743: 5, H0484: 4, H0483: 4, H0059: 4, L0764: 4, L0771: 4, L0662: 4, S0330: 4, H0696: 4, H0615: 3, H0688: 3, H0040: 3, L0763: 3, L0364: 3, L0774: 3, L0663: 3, H0683: 3, H0670: 3, H0672: 3, L0750: 3, L0731: 3, S0434: 3, L0601: 3, H0663: 2, H0549: 2, H0046: 2, H0014: 2, H0124: 2, H0494: 2, S0450: 2, L0374: 2, L0765: 2, L0804: 2, L0378: 2, L0805: 2, L0653: 2, L0529: 2, H0593: 2, H0658: 2, S0152: 2, L0439: 2, L0779: 2, H0294: 1, S0430: 1, H0675: 1, H0676: 1, H0749: 1, H0370: 1, H0587: 1, H0642: 1, T0109: 1, H0204: 1, L0040: 1, L0738: 1, H0620: 1, H0024: 1, H0356: 1, H0375: 1, L0194: 1, T0023: 1, L0483: 1, H0033: 1, H0424: 1, H0644: 1, H0264: 1, H0488: 1, H0413: 1, H0334: 1, S0352: 1, S0306: 1, H0647: 1, H0646: 1, H0652: 1, L0373: 1, L0646: 1, L0648: 1, L0794: 1, L0803: 1, L0375: 1, L0376: 1, L0806: 1, L0658: 1, L0382: 1, L0528: 1, L5623: 1, L0664: 1, S0374: 1, H0690: 1, H0682: 1, H0648: 1, S0328: 1, H0478: 1, S0432: 1, L0744: 1, L0747: 1, L0749: 1, L0755: 1, L0759: 1, H0707: 1, L0596: 1, H0543: 1, S0462: 1 and H0352: 1. 7 HDTAW95 412472 38 AR219: 109, AR218: 78, AR282: 11, AR3164, AR2993, AR3132, AR3002, AR2402, AR0391, AR0551 S0360: 9, L0794: 8, L0659: 6, S0436: 6, H0622: 5, S0126: 4, H0170: 3, H0587: 3, S0422: 3, L0800: 3, L0649: 3, S0390: 3, S0028: 3, L0777: 3, L0757: 3, H0662: 2, H0586: 2, H0013: 2, H0150: 2, H0551: 2, L0770: 2, L0761: 2, L0773: 2, H0682: 2, H0648: 2, S0406: 2, L0743: 2, L0751: 2, L0779: 2, S0026: 2, S0194: 2, H0506: 2, H0624: 1, S0212: 1, S0356: 1, S0354: 1, S0376: 1, H0643: 1, H0486: 1, H0427: 1, H0156: 1, L0021: 1, L0105: 1, H0031: 1, H0628: 1, H0316: 1, H0090: 1, H0412: 1, S0450: 1, S0440: 1, H0647: 1, L0763: 1, L0372: 1, L0646: 1, L0771: 1, L0648: 1, L0767: 1, L0768: 1, L0805: 1, L0653: 1, L0606: 1, L0789: 1, L4501: 1, H0144: 1, H0762: 1, H0547: 1, H0690: 1, H0684: 1, H0435: 1, S0152: 1, S0332: 1, H0626: 1, S0037: 1, S3014: 1, L0747: 1, L0480: 1, S0192: 1 and S0196: 1. 8 HEMCV19 423219 46 8 HEMCV19 1352162 48 AR277: 50, AR283: 34, AR219: 33, AR096: 32, AR240: 32, AR218: 31, AR316: 30, AR089: 23, AR282: 23, AR039: 23, AR104: 23, AR313: 23, AR299: 21, AR300: 20, AR185: 20, AR055: 17, AR060: 17 H0617: 7, S0114: 5, H0486: 5, H0069: 5, S0344: 5, L0794: 5, H0436: 5, L0748: 5, H0341: 4, H0255: 4, L0744: 4, S0436: 4, H0295: 3, H0294: 3, T0049: 3, H0661: 3, S0356: 3, S0476: 3, H0581: 3, H0271: 3, H0038: 3, H0087: 3, H0529: 3, L0364: 3, L0766: 3, S0126: 3, L0747: 3, L0749: 3, H0423: 3, H0740: 2, H0657: 2, S0376: 2, H0497: 2, H0635: 2, H0616: 2, S0440: 2, S0144: 2, S0142: 2, L0771: 2, L0378: 2, L0806: 2, L0518: 2, H0658: 2, S0044: 2, S3014: 2, S0027: 2, L0754: 2, L0777: 2, L0731: 2, H0445: 2, L0588: 2, L0599: 2, S0424: 2, L2852: 2, H0556: 1, T0002: 1, S0040: 1, H0717: 1, L2865: 1, H0656: 1, L2919: 1, H0381: 1, H0402: 1, H0305: 1, S0360: 1, H0580: 1, H0722: 1, H0728: 1, S0046: 1, H0747: 1, S0278: 1, H0549: 1, H0586: 1, H0333: 1, T0060: 1, L0021: 1, H0575: 1, T0048: 1, H0318: 1, H0421: 1, H0263: 1, H0597: 1, H0530: 1, H0545: 1, H0041: 1, S0388: 1, H0266: 1, S0334: 1, H0688: 1, L0483: 1, H0424: 1, H0644: 1, H0628: 1, H0182: 1, H0316: 1, H0090: 1, H0591: 1, H0063: 1, H0551: 1, H0264: 1, H0412: 1, H0100: 1, H0280: 1, S0426: 1, H0743: 1, L0369: 1, L0520: 1, L4497: 1, L0770: 1, L0769: 1, L3904: 1, L0761: 1, L0772: 1, L0800: 1, L0662: 1, L5569: 1, L0381: 1, L0775: 1, L0805: 1, L0654: 1, L0776: 1, L0655: 1, L0657: 1, L0656: 1, L0382: 1, L0809: 1, L0543: 1, L0791: 1, L0666: 1, L0664: 1, H0697: 1, H0699: 1, H0689: 1, H0690: 1, H0660: 1, H0518: 1, H0521: 1, H0696: 1, S0406: 1, H0576: 1, S3012: 1, L0743: 1, L0780: 1, L0757: 1, L0759: 1, S0031: 1, L0593: 1, L0595: 1, H0542: 1, H0543: 1, H0422: 1 and H0506: 1. 8 HEMCV19 1352161 50 9 HETBX14 806447 61 AR277: 75, AR269: 27, AR163: 22, AR165: 22, AR162: 21, AR161: 21, AR164: 21, AR089: 21, AR282: 21, AR166: 20, AR173: 17, AR243: 16, AR183: 16, AR188: 16, AR182: 15, AR268: 14, AR060: 14, AR196: 14, AR290: 14, AR238: 13, AR270: 12, AR240: 12, AR246: 12, AR291: 12, AR193: 11, AR300: 11, AR176: 11, AR245: 11, AR175: 11, AR180: 11, AR267: 10, AR191: 10, AR205: 10, AR316: 10, AR254: 10, AR250: 10, AR242: 10, AR255: 10, AR2339, AR2479, AR0969, AR2979, AR2379, AR1819, AR0398, AR1988, AR2318, AR2968, AR2108, AR1978, AR1898, AR2858, AR2758, AR2138, AR2347, AR0617, AR2397, AR1747, AR2667, AR2837, AR2007, AR3137, AR2017, AR1797, AR1777, AR1787, AR2327, AR2727, AR2717, AR2957, AR3087, AR2197, AR2877, AR2537, AR3127, AR2267, AR2276, AR2366, AR2886, AR2036, AR2996, AR2286, AR2616, AR1926, AR1856, AR2746, AR2296, AR1996, AR0536, AR1906, AR1956, AR2936, AR2606, AR2116, AR2576, AR0335, AR3115, AR2125, AR2165, AR2645, AR2255, AR3095, AR2155, AR1695, AR2895, AR2865, AR2185, AR1685, AR2235, AR2945, AR2175, AR2585, AR2044, AR2354, AR2244, AR2524, AR1714, AR2624, AR2304, AR0554, AR1704, AR2143, AR1043, AR1723, AR2633, AR2563, AR2213, AR2072, AR2222 L0751: 6, L0809: 5, S0328: 5, H0046: 4, L0758: 4, L0764: 3, L0665: 2, L0747: 2, H0592: 1, H0486: 1, H0263: 1, H0598: 1, H0063: 1, H0646: 1, L0662: 1, L0657: 1, L0543: 1, L5622: 1, L0793: 1, L0666: 1, L2654: 1, H0658: 1, S0330: 1 and S0044: 1. 9 HETBX14 422659 63 10 HLHSK94 1307727 72 AR283: 613, AR104: 327, AR219: 244, AR218: 219, AR055: 199, AR316: 176, AR313: 160, AR089: 107, AR060: 106, AR039: 106, AR096: 99, AR299: 86, AR185: 65, AR240: 56, AR282: 51, AR300: 46, AR277: 26, AR2524, AR2463, AR2233, AR2353, AR2533, AR2663, AR1703, AR1693, AR2302, AR2432, AR2622, AR3122, AR2142, AR3092, AR2612, AR1972, AR2742, AR1932, AR2552, AR0532, AR2972, AR2252, AR1682, AR2722, AR1811, AR2171, AR2871, AR2571, AR2051, AR2911, AR0331, AR1911, AR1901, AR2361, AR1751 H0046: 44, H0135: 10, H0539: 10, L0455: 7, S0010: 3, L0456: 3, L0750: 3, L0663: 2, L0746: 2, L0747: 2, L0779: 2, L0777: 2, H0624: 1, S0116: 1, H0208: 1, L0717: 1, H0549: 1, H0333: 1, H0013: 1, L0157: 1, T0006: 1, H0652: 1, L0666: 1, H0144: 1, L3811: 1, S0328: 1, H0696: 1 and L0439: 1. 10 HLHSK94 422828 74 11 HLHFP03 460467 76 AR194: 6, AR186: 6, AR169: 6, AR170: 5, AR202: 5, AR060: 5, AR206: 5, AR184: 5, AR176: 5, AR273: 4, AR249: 4, AR248: 4, AR223: 4, AR161: 4, AR162: 4, AR251: 4, AR163: 4, AR061: 4, AR244: 4, AR052: 4, AR055: 4, AR310: 4, AR282: 4, AR053: 4, AR267: 4, AR253: 3, AR235: 3, AR183: 3, AR269: 3, AR182: 3, AR312: 3, AR204: 3, AR266: 3, AR192: 3, AR246: 3, AR275: 3, AR185: 3, AR270: 3, AR283: 3, AR089: 3, AR298: 3, AR295: 3, AR039: 3, AR241: 3, AR271: 3, AR309: 3, AR181: 3, AR166: 3, AR291: 3, AR263: 3, AR257: 3, AR217: 3, AR316: 3, AR289: 3, AR299: 3, AR296: 3, AR033: 3, AR238: 3, AR292: 3, AR205: 2, AR247: 2, AR313: 2, AR104: 2, AR193: 2, AR231: 2, AR213: 2, AR268: 2, AR168: 2, AR284: 2, AR262: 2, AR277: 2, AR237: 2, AR212: 2, AR243: 2, AR274: 2, AR297: 2, AR286: 2, AR228: 2, AR240: 2, AR233: 2, AR272: 2, AR285: 2, AR300: 2, AR165: 2, AR229: 2, AR226: 2, AR096: 2, AR293: 2, AR255: 2, AR294: 2, AR191: 2, AR290: 2, AR164: 2, AR172: 2, AR264: 2, AR227: 2, AR174: 2, AR287: 2, AR198: 2, AR265: 2, AR232: 2, AR171: 2, AR216: 2, AR177: 2, AR311: 1, AR234: 1, AR175: 1, AR239: 1, AR203: 1, AR236: 1, AR230: 1, AR196: 1, AR261: 1, AR260: 1, AR259: 1, AR201: 1, AR189: 1, AR179: 1, L0742: 4 and H0024: 1. 11 HLHFP03 460467 78 AR1946, AR1866, AR1696, AR1705, AR2025, AR0605, AR2065, AR1845, AR1765, AR2734, AR2494, AR2484, AR2234, AR1614, AR0554, AR1624, AR2514, AR1634, AR0614, AR2824, AR2444, AR0524, AR3104, AR0534, AR2674, AR2533, AR2353, AR1833, AR2693, AR1823, AR3123, AR2043, AR2663, AR1923, AR2463, AR2753, AR2703, AR1043, AR1853, AR2983, AR0893, AR2953, AR2413, AR2713, AR3093, AR1813, AR1663, AR2913, AR2633, AR2573, AR2173, AR2893, AR2963, AR0333, AR2383, AR2833, AR2773, AR2923, AR2052, AR2472, AR2992, AR1932, AR2312, AR2132, AR2682, AR1682, AR2842, AR2622, AR2372, AR2122, AR2432, AR2742, AR2972, AR3002, AR2862, AR2282, AR2402, AR2332, AR2722, AR2852, AR3162, AR1652, AR2292, AR0962, AR2262, AR2932, AR3132, AR2552, AR2942, AR1912, AR2902, AR1642, AR1722, AR2642, AR2272, AR1742, AR0392, AR2872, AR1982, AR2652, AR2322, AR1712, AR2162, AR1772, AR3111, AR2341, AR1751, AR2391, AR2031, AR2361, AR2301, AR2181, AR1961, AR2611, AR2601, AR2591, AR2011, AR1891, AR1791 L0742: 4 and H0024: 1. 12 HHTLF25 461438 83 AR251: 168, AR248: 141, AR249: 139, AR265: 60, AR253: 50, AR263: 41, AR096: 37, AR244: 32, AR268: 26, AR264: 24, AR290: 20, AR246: 18, AR240: 17, AR177: 16, AR267: 14, AR183: 14, AR270: 13, AR229: 13, AR184: 12, AR055: 11, AR269: 10, AR274: 9, AR194: 8, AR175: 8, AR247: 7, AR202: 7, AR316: 7, AR234: 7, AR033: 6, AR180: 6, AR198: 6, AR271: 6, AR182: 6, AR238: 6, AR299: 5, AR206: 5, AR190: 5, AR205: 5, AR313: 5, AR188: 5, AR275: 5, AR272: 5, AR061: 5, AR196: 5, AR284: 5, AR241: 5, AR273: 5, AR173: 5, AR189: 5, AR203: 5, AR199: 5, AR237: 5, AR179: 4, AR200: 4, AR039: 4, AR172: 4, AR191: 4, AR298: 4, AR192: 4, AR181: 4, AR291: 4, AR282: 4, AR289: 4, AR176: 4, AR224: 4, AR292: 4, AR186: 3, AR226: 3, AR174: 3, AR300: 3, AR165: 3, AR161: 3, AR162: 3, AR266: 3, AR285: 3, AR163: 3, AR164: 3, AR231: 3, AR052: 3, AR215: 3, AR295: 3, AR212: 3, AR243: 3, AR309: 3, AR221: 3, AR166: 3, AR169: 3, AR296: 3, AR232: 2, AR053: 2, AR185: 2, AR223: 2, AR225: 2, AR089: 2, AR104: 2, AR277: 2, AR233: 2, AR213: 2, AR308: 2, AR286: 2, AR256: 2, AR257: 2, AR310: 2, AR283: 2, AR235: 2, AR217: 2, AR227: 2, AR204: 2, AR288: 2, AR195: 2, AR281: 2, AR293: 1, AR312: 1, AR214: 1, AR261: 1, AR294: 1, AR236: 1, AR216: 1, AR193: 1, AR259: 1, AR230: 1, S0144: 10, L0775: 10, S0278: 6, H0638: 5, H0641: 5, L0438: 5, H0580: 4, S0142: 4, H0521: 4, H0522: 4, L0747: 4, H0441: 3, S0388: 3, S0428: 3, H0402: 2, S0358: 2, S0408: 2, S0140: 2, H0392: 2, H0438: 2, H0086: 2, L0520: 2, L0763: 2, L0770: 2, L0772: 2, L0771: 2, L0774: 2, L0776: 2, L0526: 2, H0658: 2, L0743: 2, L0439: 2, L0751: 2, L0754: 2, L0756: 2, L0605: 2, S0134: 1, S0116: 1, H0662: 1, S0444: 1, S0360: 1, H0637: 1, S0045: 1, S0222: 1, S6014: 1, H0455: 1, H0592: 1, H0250: 1, H0069: 1, H0575: 1, T0082: 1, H0036: 1, H0581: 1, H0457: 1, S0050: 1, S0051: 1, H0399: 1, H0354: 1, H0594: 1, H0247: 1, H0271: 1, L0055: 1, S0036: 1, S0038: 1, S0438: 1, H0646: 1, L0769: 1, L0764: 1, L0375: 1, L0787: 1, S0053: 1, S0374: 1, H0682: 1, H0648: 1, H0710: 1, L0744: 1, L0755: 1, L0731: 1, L0758: 1, L0599: 1, L0603: 1, H0423: 1 and H0352: 1. 12 HHTLF25 461438 83 AR251: 168, AR248: 141, AR249: 139, AR265: 60, AR253: 50, AR263: 41, AR244: 32, AR096: 32, AR268: 26, AR264: 24, AR290: 20, AR246: 18, AR240: 17, AR177: 16, AR267: 14, AR183: 14, AR270: 13, AR229: 13, AR184: 12, AR269: 10, AR2749, AR1948, AR1758, AR3167, AR2477, AR2027, AR3137, AR2347, AR0556, AR2996, AR0336, AR1806, AR1986, AR2716, AR1826, AR2386, AR2065, AR1905, AR2055, AR1885, AR2755, AR2725, AR0615, AR1965, AR2845, AR2415, AR2735, AR1735, AR1895, AR2035, AR1995, AR2375, AR1794, AR0394, AR2004, AR1724, AR1914, AR2984, AR1924, AR1814, AR2914, AR1044, AR2894, AR1764, AR2244, AR2924, AR1863, AR2823, AR2263, AR1743, AR3003, AR1653, AR1613, AR1623, AR2663, AR2853, AR1633, AR1643, AR2313, AR1853, AR0523, AR2153, AR2953, AR2123, AR2433, AR3093, AR2213, AR1663, AR1693, AR2963, AR2322, AR0532, AR2232, AR2252, AR0892, AR2772, AR2332, AR2132, AR3082, AR2862, AR2562, AR2572, AR2832, AR3102, AR2352, AR2172, AR2272, AR2042, AR2882, AR1952, AR2812, AR2931, AR3121, AR2141, AR2611, AR2941, AR2361, AR2161, AR1931, AR2591, AR2301 S0144: 10, L0775: 10, S0278: 6, H0638: 5, H0580: 5, H0641: 5, L0438: 5, H0521: 5, H0740: 4, H0392: 4, H0522: 4, L0747: 4, S0408: 3, H0749: 3, H0441: 3, H0438: 3, S0388: 3, S0428: 3, H0658: 3, H0402: 2, S0358: 2, S0444: 2, S0140: 2, H0747: 2, H0086: 2, S0142: 2, L0520: 2, L0763: 2, L0770: 2, L0772: 2, L0771: 2, L0774: 2, L0776: 2, L0526: 2, L0743: 2, L0439: 2, L0751: 2, L0754: 2, L0756: 2, L0605: 2, S0116: 1, H0662: 1, S0360: 1, L3646: 1, H0637: 1, S0045: 1, S0222: 1, S6014: 1, H0455: 1, H0592: 1, H0250: 1, H0069: 1, H0575: 1, T0082: 1, H0036: 1, H0581: 1, H0457: 1, S0050: 1, S0051: 1, H0399: 1, H0354: 1, H0594: 1, H0247: 1, H0271: 1, L0055: 1, S0036: 1, S0038: 1, S0438: 1, H0646: 1, L0769: 1, L0764: 1, L0375: 1, L0787: 1, S0053: 1, S0374: 1, H0682: 1, H0648: 1, H0710: 1, S0152: 1, H0727: 1, L0744: 1, L0755: 1, L0731: 1, L0758: 1, L0599: 1, L0603: 1, H0423: 1 and H0352: 1. 13 HTADX17 457172 90 AR227: 8, AR293: 7, AR176: 7, AR233: 7, AR229: 7, AR232: 6, AR179: 6, AR182: 6, AR237: 6, AR296: 6, AR266: 6, AR060: 5, AR294: 5, AR287: 5, AR252: 4, AR185: 4, AR286: 4, AR239: 4, AR255: 4, AR200: 4, AR230: 4, AR271: 4, AR221: 3, AR289: 3, AR282: 3, AR297: 3, AR162: 3, AR089: 3, AR291: 3, AR290: 3, AR275: 3, AR257: 3, AR096: 3, AR316: 3, AR253: 2, AR270: 2, AR175: 2, AR242: 2, AR228: 2, AR061: 2, AR262: 2, AR269: 2, AR172: 2, AR161: 2, AR283: 2, AR168: 2, AR183: 2, AR181: 2, AR205: 2, AR300: 2, AR231: 2, AR177: 2, AR267: 2, AR104: 2, AR261: 2, AR033: 2, AR264: 2, AR225: 2, AR277: 2, AR195: 2, AR215: 2, AR214: 2, AR238: 2, AR173: 2, AR197: 2, AR201: 2, AR268: 2, AR246: 2, AR299: 2, AR188: 2, AR313: 2, AR288: 2, AR216: 2, AR285: 2, AR234: 1, AR309: 1, AR169: 1, AR240: 1, AR224: 1, AR174: 1, AR190: 1, AR165: 1, AR274: 1, AR039: 1, AR178: 1, AR217: 1, AR260: 1, AR196: 1, AR191: 1, AR204: 1, AR311: 1, AR055: 1, AR166: 1, AR222: 1, AR210: 1, AR171: 1, AR199: 1, AR258: 1, H0069: 5, H0634: 1 and L0772: 1. 13 HTADX17 753289 92 AR2278, AR2937, AR1767, AR2337, AR2297, AR2326, AR1796, AR1826, AR2376, AR2966, AR2666, AR0605, AR2945, AR0555, AR2875, AR2524, AR2864, AR1854, AR2394, AR2554, AR2004, AR2304, AR2714, AR2213, AR2893, AR2823, AR2973, AR1623, AR0893, AR2913, AR2903, AR2753, AR2573, AR0963, AR2532, AR2702, AR1752, AR2422, AR2282, AR3162, AR0612, AR2622, AR2692, AR1722, AR1612, AR3002, AR1682, AR2832, AR1832, AR1812, AR2052, AR2312, AR1772, AR2672, AR2612, AR0332, AR2642, AR2252, AR1952, AR2772, AR2152, AR3132, AR2142, AR2382, AR1732, AR1972, AR0392, AR2012, AR2682, AR1042, AR2462, AR2182, AR1882, AR2882, AR2162, AR2992, AR2852, AR2341, AR3091, AR1691, AR2401, AR2241, AR1741, AR1901, AR1651, AR2741, AR1781, AR2171, AR2601, AR1961, AR1911, AR2041, AR3111, AR1661, AR2221, AR2101, AR1711, AR1991, AR2581 H0069: 5, H0634: 1 and L0772: 1. 13 HTADX17 457172 94 14 HJACG02 509948 102 AR207: 37, AR195: 33, AR283: 32, AR263: 32, AR264: 29, AR223: 28, AR214: 28, AR089: 28, AR277: 27, AR222: 27, AR309: 27, AR311: 27, AR212: 26, AR316: 26, AR169: 26, AR224: 24, AR096: 24, AR055: 24, AR197: 23, AR213: 23, AR282: 22, AR104: 22, AR245: 22, AR171: 22, AR218: 22, AR162: 22, AR192: 21, AR217: 21, AR161: 21, AR193: 21, AR163: 20, AR308: 20, AR165: 20, AR168: 20, AR216: 20, AR170: 20, AR164: 20, AR235: 19, AR172: 19, AR053: 19, AR166: 19, AR060: 19, AR219: 19, AR242: 19, AR271: 19, AR299: 19, AR210: 19, AR039: 19, AR033: 19, AR240: 18, AR225: 18, AR313: 18, AR312: 18, AR201: 18, AR221: 17, AR261: 17, AR198: 17, AR246: 17, AR288: 17, AR252: 17, AR295: 17, AR176: 16, AR177: 16, AR215: 15, AR297: 15, AR253: 15, AR205: 15, AR270: 15, AR196: 15, AR185: 15, AR275: 15, AR286: 15, AR285: 14, AR260: 14, AR287: 14, AR233: 14, AR236: 14, AR183: 14, AR227: 13, AR175: 13, AR211: 13, AR300: 13, AR250: 13, AR294: 13, AR181: 13, AR274: 13, AR272: 13, AR229: 13, AR174: 12, AR256: 12, AR182: 12, AR234: 12, AR204: 12, AR269: 12, AR228: 12, AR293: 12, AR178: 12, AR226: 12, AR268: 11, AR266: 11, AR173: 11, AR262: 11, AR200: 11, AR243: 11, AR199: 11,AR258: 11,AR231: 11,AR291: 11, AR180: 11, AR289: 11, AR247: 11, AR239: 10, AR257: 10, AR267: 10, AR255: 10, AR188: 10, AR254: 10, AR203: 10, AR232: 10, AR238: 9, AR191: 9, AR189: 9, AR190: 9, AR061: 9, AR230: 9, AR296: 9, AR179: 9, AR290: 8, AR237: 7, S0442: 4, L0764: 4, S0408: 3, H0306: 2, H0263: 2, H0596: 2, L0800: 2, L0755: 2, S0116: 1, S0358: 1, H0489: 1, H0597: 1, T0041: 1 and L0772: 1. 14 HJACG02 1307789 104 AR207: 37, AR195: 33, AR283: 32, AR263: 32, AR264: 29, AR223: 28, AR214: 28, AR089: 28, AR277: 27, AR222: 27, AR309: 27, AR311: 27, AR212: 26, AR169: 26, AR316: 25, AR224: 24, AR096: 24, AR055: 24, AR197: 23, AR213: 23, AR282: 22, AR104: 22, AR245: 22, AR171: 22, AR218: 22, AR162: 22, AR192: 21, AR217: 21, AR161: 21, AR193: 21, AR163: 20, AR308: 20, AR165: 20, AR168: 20, AR216: 20, AR170: 20, AR164: 20, AR235: 19, AR172: 19, AR053: 19, AR166: 19, AR060: 19, AR219: 19, AR242: 19, AR271: 19, AR299: 19, AR210: 19, AR039: 19, AR033: 19, AR240: 18, AR225: 18, AR313: 18, AR312: 18, AR201: 18, AR221: 17, AR261: 17, AR198: 17, AR246: 17, AR288: 17, AR252: 17, AR295: 17, AR176: 16, AR177: 16, AR215: 15, AR297: 15, AR253: 15, AR205: 15, AR270: 15, AR196: 15, AR185: 15, AR275: 15, AR286: 15, AR285: 14, AR260: 14, AR287: 14, AR233: 14, AR236: 14, AR183: 14, AR227: 13, AR175: 13, AR211: 13, AR300: 13, AR250: 13, AR294: 13, AR181: 13, AR274: 13, AR272: 13, AR229: 13, AR174: 12, AR256: 12, AR182: 12, AR234: 12, AR204: 12, AR269: 12, AR228: 12, AR293: 12, AR178: 12, AR226: 12, AR268: 11, AR266: 11, AR173: 11, AR262: 11, AR200: 11, AR243: 11, AR199: 11,AR258: 11,AR231: 11,AR291: 11, AR180: 11, AR289: 11, AR247: 11, AR239: 10, AR257: 10, AR267: 10, AR255: 10, AR188: 10, AR254: 10, AR203: 10, AR232: 10, AR2389, AR1919, AR1899, AR1909, AR0619, AR2309, AR2969, AR1799, AR2908, AR2377 S0442: 4, L0764: 4, S0408: 3, H0306: 2, H0263: 2, H0596: 2, L0800: 2, L0755: 2, S0116: 1, S0358: 1, H0489: 1, H0597: 1, T0041: 1 and L0772: 1. 15 HKGAJ54 498303 107 AR218: 7, AR219: 6, AR242: 6, AR315: 6, AR225: 5, AR248: 4, AR244: 4, AR273: 4, AR263: 4, AR310: 4, AR280: 4, AR214: 4, AR282: 4, AR314: 3, AR221: 3, AR170: 3, AR311: 3, AR052: 3, AR213: 3, AR162: 3, AR217: 3, AR281: 3, AR243: 3, AR253: 2, AR270: 2, AR265: 2, AR186: 2, AR277: 2, AR055: 2, AR207: 2, AR104: 2, AR257: 2, AR316: 2, AR212: 2, AR194: 2, AR312: 2, AR206: 2, AR089: 2, AR195: 2, AR283: 2, AR171: 2, AR193: 1, AR240: 1, AR284: 1, AR199: 1, AR039: 1, AR163: 1, AR053: 1, AR183: 1, AR288: 1, AR033: 1, AR309: 1, AR060: 1, AR096: 1, AR236: 1, AR188: 1, AR266: 1, AR216: 1, AR161: 1, AR201: 1, AR205: 1, S0474: 2, S0400: 1, H0661: 1, S0045: 1, T0039: 1, S0003: 1, H0412: 1, H0056: 1, H0494: 1, H0646: 1, H0538: 1, S0422: 1, L0533: 1, H0539: 1, L0740: 1, L0777: 1, S0436: 1 and L0588: 1. 15 HKGAJ54 1300770 109 AR2187, AR2196, AR2426, AR3156, AR2255, AR2484, AR2444, AR2734, AR2634, AR3104, AR2804, AR2144, AR2824, AR3143, AR2213, AR1703, AR3113, AR0523, AR2133, AR1623, AR2173, AR2813, AR2433, AR2532, AR2702, AR2652, AR1862, AR2772, AR0552, AR3162, AR2072, AR1042, AR2572, AR2122, AR1942, AR3122, AR2062, AR0892, AR1952, AR2832, AR1712, AR1931, AR2401, AR2841, AR1991, AR0391, AR1631, AR0531, AR1831, AR2881, AR0331, AR3091, AR0601, AR0961, AR2361, AR1881, AR2661, AR2161, AR1611, AR2011, AR2051 S0474: 2, S0400: 1, H0661: 1, S0045: 1, T0039: 1, S0003: 1, H0412: 1, H0056: 1, H0494: 1, H0646: 1, H0773: 1, H0538: 1, S0422: 1, L0533: 1, H0539: 1, L0740: 1, L0777: 1, S0436: 1 and L0588: 1. 16 HSVAK93 597462 116 16 HE8CH92 609866 118 AR290: 19, AR268: 11, AR2679, AR3129, AR3098, AR2518, AR2707, AR3107, AR0537, AR2826, AR2666, AR3136, AR0526, AR2656, AR2635, AR0615, AR2695, AR2935, AR0605, AR0335, AR1825, AR2925, AR2955, AR2864, AR2484, AR0554, AR2384, AR2414, AR2534, AR2984, AR2134, AR0394, AR2474, AR2854, AR2894, AR1754, AR2914, AR2294, AR2944, AR2964, AR0893, AR2993, AR1773, AR2493, AR0963, AR2843, AR2563, AR2373, AR2333, AR3163, AR2343, AR2263, AR3003, AR1843, AR2593, AR1853, AR2313, AR2833, AR1832, AR2402, AR2322, AR2772, AR1042, AR1862, AR2192, AR2272, AR2182, AR2582, AR1792 L0769: 7, L0754: 7, H0657: 4, L0764: 4, L0751: 4, L0752: 4, S0408: 3, S0222: 3, H0327: 3, H0046: 3, H0056: 3, L0756: 3, S0282: 2, S0356: 2, S0444: 2, H0580: 2, L3653: 2, H0250: 2, H0318: 2, S0474: 2, H0622: 2, H0625: 2, L0803: 2, L0809: 2, L0666: 2, L2654: 2, H0648: 2, H0672: 2, S0380: 2, S0146: 2, L0748: 2, L0745: 2, L0747: 2, L0777: 2, L0731: 2, L0600: 2, H0556: 1, H0740: 1, S0116: 1, H0305: 1, H0125: 1, S0410: 1, H0637: 1, S0476: 1, H0393: 1, H0586: 1, H0643: 1, H0486: 1, H0013: 1, H0635: 1, H0599: 1, H0575: 1, S0346: 1, H0052: 1, H0309: 1, H0050: 1, H0024: 1, H0266: 1, H0271: 1, H0188: 1, H0286: 1, H0615: 1, H0124: 1, H0135: 1, H0634: 1, H0264: 1, H0413: 1, T0042: 1, H0396: 1, H0132: 1, H0641: 1, H0652: 1, S0422: 1, L0639: 1, L0646: 1, L0771: 1, L0773: 1, L0662: 1, L0766: 1, L0774: 1, L0776: 1, L0655: 1, L0807: 1, L0659: 1, L0367: 1, L2261: 1, L2440: 1, L2464: 1, H0726: 1, H0547: 1, H0670: 1, H0521: 1, H0696: 1, S0404: 1, S0406: 1, L0740: 1, L0749: 1, L0779: 1, L0755: 1, L0759: 1, H0445: 1, S0434: 1, S0026: 1, H0653: 1, H0667: 1, S0242: 1, S0412: 1, H0506: 1 and : 1. 16 HSVAK93 1352228 120 AR205: 32, AR269: 30, AR275: 23, AR234: 22, AR281: 21, AR299: 20, AR315: 17, AR194: 17, AR231: 17, AR271: 17, AR274: 16, AR282: 16, AR177: 16, AR202: 15, AR283: 15, AR266: 15, AR246: 15, AR310: 14, AR280: 14, AR273: 14, AR265: 14, AR033: 14, AR244: 13, AR268: 13, AR206: 13, AR204: 13, AR053: 13, AR175: 13, AR289: 12, AR052: 12, AR192: 12, AR309: 12, AR314: 12, AR237: 12, AR039: 12, AR247: 12, AR198: 12, AR184: 11, AR238: 11, AR295: 11, AR284: 11, AR243: 11, AR270: 11, AR213: 11, AR241: 11, AR277: 11, AR291: 10, AR055: 10, AR312: 10, AR183: 10, AR285: 10, AR256: 10, AR290: 10, AR267: 10, AR263: 10, AR292: 10, AR2869, AR2969, AR2519, AR2989, AR1859, AR2408, AR1868, AR0618, AR3168, AR3008, AR2938, AR2278, AR2328, AR0898, AR2947, AR1827, AR3137, AR2297, AR2267, AR1047, AR2187, AR0966, AR2336, AR2196, AR2486, AR2535, AR2595, AR1795, AR2495, AR2585, AR0605 L0438: 4, L0439: 4, S0116: 3, L0749: 3, H0663: 2, H0309: 2, H0032: 2, H0616: 2, L0646: 2, L0774: 2, S0428: 2, L0748: 2, L0777: 2, H0445: 2, H0556: 1, S0134: 1, H0586: 1, H0632: 1, H0013: 1, H0427: 1, H0575: 1, H0318: 1, H0421: 1, H0050: 1, S0050: 1, H0051: 1, H0598: 1, H0413: 1, H0494: 1, H0396: 1, S0344: 1, L0769: 1, L0772: 1, L0666: 1, L0664: 1, L0665: 1, S0216: 1, H0726: 1, H0435: 1, S0378: 1, H0521: 1, S0146: 1, L0779: 1, L0755: 1 and L0731: 1. 17 HSDEK49 625998 123 17 HSDEK49 1352253 125 AR290: 45, AR268: 37, AR240: 23, AR267: 22, AR269: 16, AR270: 14, AR234: 10, AR055: 10, AR238: 10, AR1849, AR2928, AR2918, AR1798, AR1838, AR2847, AR1777, AR1826, AR0606, AR2995, AR2955, AR2855, AR2445, AR2935, AR1755, AR0964, AR1853, AR2293, AR2493, AR2963, AR3163, AR2313, AR2983, AR2893, AR1043, AR2373, AR2862, AR0892, AR2262, AR2042, AR2662, AR2822, AR2942, AR2272, AR3132, AR2472, AR3002, AR2332, AR2482, AR2592, AR2752, AR2562, AR0391, AR0331, AR2771, AR2631, AR0611, AR2581, AR2321, AR2711, AR2831, AR3101 H0031: 7, L0439: 7, L0754: 7, L3388: 6, L0731: 6, S0002: 5, H0580: 4, H0575: 3, H0309: 3, L0438: 3, H0555: 3, L0758: 3, S0360: 2, L3649: 2, H0553: 2, S0344: 2, S0426: 2, L0775: 2, S0330: 2, L0747: 2, L0779: 2, S0260: 2, L0599: 2, L0603: 2, H0739: 1, H0170: 1, S0116: 1, S0354: 1, S0444: 1, L3645: 1, H0270: 1, S0280: 1, H0590: 1, H0581: 1, H0251: 1, H0014: 1, H0355: 1, H0030: 1, H0644: 1, H0674: 1, H0090: 1, H0063: 1, S0142: 1, L0770: 1, L0769: 1, L0651: 1, L0776: 1, L0659: 1, L0519: 1, L0664: 1, H0682: 1, L0749: 1, L0752: 1, S0031: 1 and H0506: 1. 18 HWBAO62 838164 127 AR252: 43, AR264: 25, AR311: 20, AR308: 19, AR245: 16, AR254: 15, AR250: 15, AR246: 14, AR309: 13, AR195: 13, AR197: 13, AR201: 13, AR263: 13, AR212: 12, AR272: 11, AR193: 10, AR1749, AR2059, AR0539, AR2079, AR2438, AR2008, AR2258, AR1988, AR2538, AR2238, AR1888, AR2228, AR2247, AR3127, AR1897, AR1707, AR1637, AR2137, AR1927, AR2216, AR1616, AR1966, AR1626, AR1916, AR1656, AR2426, AR1646, AR1736, AR1805, AR1785, AR1695, AR2115, AR2405, AR2105, AR2745, AR1905, AR1725, AR2885, AR1665, AR2035, AR1815, AR1995, AR2165, AR2905, AR2575, AR2185, AR2615, AR1845, AR2695, AR2044, AR2974, AR1684, AR1764, AR2144, AR2304, AR1834, AR2874, AR2624, AR2354, AR2554, AR2684, AR2704, AR2674, AR2664, AR1793, AR2173, AR2193, AR1853, AR1713, AR1773, AR2963, AR1753, AR2823, AR3133, AR0393, AR0963, AR2153, AR2753, AR2363, AR1823, AR0893, AR2343, AR2473, AR3163, AR2953, AR0333, AR2313, AR2913, AR2383, AR2653, AR2392, AR2892, AR3002, AR0522, AR2772, AR2852, AR2712, AR2332, AR2292, AR2282, AR2582, AR2372, AR2322, AR0602, AR2932, AR2942, AR2602, AR2862, AR2992, AR2262, AR2062, AR3102, AR0612, AR2732, AR1862, AR2921, AR2561, AR1041, AR2811, AR2271, AR2831 H0580: 1 and H0427: 1. 18 HWBAO62 625914 129 19 HWHGU54 695695 131 AR223: 5, AR169: 4, AR171: 4, AR221: 4, AR224: 4, AR264: 4, AR214: 4, AR261: 3, AR235: 3, AR263: 3, AR195: 3, AR225: 3, AR311: 3, AR168: 3, AR216: 3, AR222: 3, AR238: 3, AR183: 3, AR172: 3, AR297: 2, AR212: 2, AR162: 2, AR161: 2, AR251: 2, AR170: 2, AR217: 2, AR269: 2, AR207: 2, AR272: 2, AR228: 2, AR288: 2, AR308: 2, AR237: 2, AR231: 2, AR163: 2, AR266: 2, AR312: 2, AR176: 2, AR282: 2, AR165: 2, AR257: 2, AR262: 2, AR277: 2, AR200: 2, AR196: 2, AR198: 2, AR173: 2, AR254: 2, AR213: 2, AR166: 2, AR180: 2, AR226: 2, AR181: 2, AR234: 2, AR298: 2, AR189: 2, AR096: 2, AR236: 2, AR089: 2, AR271: 2, AR197: 2, AR246: 2, AR295: 2, AR193: 2, AR239: 2, AR274: 1, AR178: 1, AR061: 1, AR227: 1, AR300: 1, AR177: 1, AR039: 1, AR164: 1, AR188: 1, AR267: 1, AR247: 1, AR287: 1, AR229: 1, AR211: 1, AR243: 1, AR201: 1, AR191: 1, AR204: 1, AR190: 1, AR179: 1, AR270: 1, AR182: 1, AR055: 1, AR230: 1, AR294: 1, AR199: 1, AR285: 1, AR291: 1, AR290: 1, AR316: 1, AR286: 1, AR296: 1, AR060: 1, AR309: 1, AR210: 1, H0586: 3 and L0777: 2. 19 HWHGU54 695695 131 AR2235, AR1694, AR1714, AR2214, AR2244, AR2644, AR2144, AR2613, AR2353, AR2633, AR1953, AR2253, AR3113, AR1683, AR2163, AR2223, AR2383, AR1833, AR1723, AR2972, AR2122, AR1622, AR1612, AR2512, AR1702, AR2172, AR2692, AR2072, AR2722, AR2282, AR2882, AR3082, AR2372, AR2312, AR1632, AR2662, AR3122, AR1762, AR2822, AR1652, AR2572, AR2622, AR2772, AR2002, AR1962, AR1982, AR1732, AR2542, AR2132, AR1662, AR1802, AR2262, AR1812, AR2342, AR2982, AR1892, AR2362, AR2712, AR1972, AR0892, AR2462, AR2952, AR1932, AR2392, AR2741, AR1781, AR0611, AR2271, AR3001, AR1771, AR1641, AR1881, AR2671, AR2471, AR0961, AR2871, AR2291, AR2111, AR2431, AR2011, AR1911, AR2041, AR1901, AR1791, AR2701, AR1821, AR2301, AR2941, AR1991, AR2851, AR2911, AR2901, AR3161, AR2861, AR2961, AR0601, AR3091, AR2101 H0586: 3 and L0777: 2. 20 HCEJQ69 1243825 134 AR273: 54, AR251: 54, AR186: 47, AR249: 31, AR052: 30, AR292: 30, AR241: 24, AR184: 24, AR061: 23, AR259: 23, AR310: 23, AR206: 22, AR194: 22, AR298: 21, AR248: 20, AR265: 18, AR274: 18, AR244: 16, AR198: 15, AR237: 15, AR243: 15, AR202: 14, AR309: 14, AR229: 14, AR033: 14, AR227: 13, AR053: 13, AR271: 13, AR204: 13, AR096: 12, AR104: 12, AR275: 12, AR192: 12, AR185: 12, AR313: 12, AR284: 11, AR296: 11, AR293: 10, AR233: 10, AR312: 10, AR282: 10, AR294: 10, AR232: 10, AR055: 10, AR283: 9, AR263: 9, AR300: 9, AR215: 9, AR266: 9, AR183: 9, AR247: 9, AR219: 9, AR256: 9, AR231: 8, AR175: 8, AR213: 8, AR267: 8, AR177: 8, AR238: 8, AR089: 8, AR218: 8, AR289: 8, AR039: 8, AR246: 7, AR277: 7, AR253: 7, AR295: 7, AR285: 7, AR299: 7, AR205: 7, AR182: 7, AR240: 7, AR286: 7, AR221: 7, AR226: 7, AR316: 6, AR234: 6, AR291: 6, AR269: 6, AR270: 6, AR060: 6, AR170: 6, AR290: 5, AR223: 5, AR179: 5, AR268: 5, AR214: 5, AR172: 5, AR224: 5, AR258: 5, AR245: 4, AR171: 4, AR216: 4, AR168: 4, AR222: 4, AR165: 4, AR254: 4, AR164: 4, AR217: 3, AR161: 3, AR225: 3, AR197: 3, AR162: 3, AR166: 3, AR163: 3, AR308: 3, AR272: 3, AR264: 3, AR257: 3, AR169: 3, AR311: 2, AR195: 2, AR212: 2, AR199: 2, AR210: 2, AR235: 2, AR180: 2, AR242: 2, AR188: 2, AR181: 2, AR288: 2, AR262: 2, AR173: 2, AR201: 2, AR178: 2, AR207: 2, AR189: 2, AR261: 2, AR196: 2, AR203: 2, AR255: 2, AR297: 2, AR236: 2, AR176: 1, AR211: 1, AR193: 1, AR287: 1, AR230: 1, AR200: 1, AR191: 1, AR174: 1, AR239: 1, AR190: 1, AR228: 1, H0052: 3, L0439: 3, H0194: 1, H0546: 1, H0615: 1, H0135: 1, H0087: 1, L0759: 1, S0436: 1 and S0456: 1. 20 HCEJQ69 1243825 134 AR273: 54, AR251: 54, AR186: 47, AR249: 31, AR052: 30, AR292: 30, AR241: 24, AR184: 24, AR061: 23, AR259: 23, AR310: 23, AR206: 22, AR194: 22, AR298: 21, AR248: 20, AR265: 18, AR274: 18, AR244: 16, AR198: 15, AR314: 15, AR237: 15, AR243: 15, AR202: 14, AR309: 14, AR229: 14, AR280: 14, AR033: 14, AR227: 13, AR053: 13, AR271: 13, AR204: 13, AR315: 13, AR104: 12, AR275: 12, AR192: 12, AR185: 12, AR313: 12, AR284: 11, AR296: 11, AR293: 10, AR233: 10, AR096: 10, AR281: 10, AR312: 10, AR282: 10, AR294: 10, AR219: 10, AR232: 10, AR0399, AR2839, AR2639, AR3009, AR2159, AR0559, AR2669, AR1839, AR2479, AR2569, AR2318, AR1758, AR2138, AR2678, AR1778, AR2388, AR2898, AR2467, AR2777, AR2537, AR0897, AR2957, AR2857, AR2997, AR2187, AR2057, AR1827, AR2407, AR2867, AR2217, AR2267, AR3166, AR2346, AR2916, AR2696, AR2706, AR0606, AR1706, AR2905, AR2235, AR1795, AR2685, AR2145, AR1725, AR2245, AR2585, AR2454, AR1714, AR2164, AR1684, AR2224, AR1654, AR2544, AR1644, AR2173, AR1613, AR2253, AR1973, AR1623, AR1663, AR1633, AR3083, AR2723, AR2643, AR2573, AR1693, AR3112, AR1952, AR2122, AR1992, AR2102, AR2352, AR1802, AR2422, AR1882, AR1812, AR2882, AR2622, AR1732, AR2012, AR1782, AR2072, AR1892, AR2612, AR1962, AR2032, AR2552, AR2972, AR2362, AR1761, AR2111, AR1931, AR2871, AR2301, AR2001, AR1911, AR1741, AR2391, AR1901, AR2281 H0052: 3, L0439: 3, H0194: 1, H0546: 1, H0615: 1, H0135: 1, H0087: 1, L3841: 1, L0759: 1, S0436: 1 and S0456: 1. 20 HCEJQ69 872582 136 20 HCEJQ69 609999 138 21 HT5GJ57 740767 159 AR213: 5, AR264: 4, AR224: 4, AR254: 4, AR253: 4, AR207: 4, AR053: 3, AR197: 3, AR161: 3, AR162: 3, AR163: 3, AR250: 3, AR221: 3, AR171: 3, AR311: 3, AR212: 3, AR165: 3, AR215: 3, AR282: 3, AR217: 2, AR309: 2, AR263: 2, AR193: 2, AR195: 2, AR089: 2, AR168: 2, AR166: 2, AR164: 2, AR312: 2, AR308: 2, AR223: 2, AR183: 2, AR267: 2, AR169: 2, AR198: 2, AR266: 2, AR033: 2, AR216: 2, AR271: 2, AR096: 1, AR210: 1, AR192: 1, AR277: 1, AR272: 1, AR240: 1, AR286: 1, AR313: 1, AR060: 1, AR180: 1, AR204: 1, AR299: 1, AR039: 1, AR246: 1, AR104: 1, AR201: 1, AR270: 1, AR205: 1, AR055: 1, AR225: 1, AR247: 1, AR258: 1, AR182: 1, AR274: 1, AR214: 1, AR287: 1, AR243: 1, H0584: 3, H0341: 3, H0271: 3, H0318: 2, H0090: 2, H0264: 2, S0052: 2, H0656: 1, H0580: 1, S0278: 1, H0431: 1, H0013: 1, H0457: 1, H0284: 1, S0003: 1, S0144: 1, S0142: 1, S0002: 1, H0702: 1, H0522: 1 and H0445: 1. 21 HT5GJ57 1299921 161 AR2135, AR2644, AR2244, AR2544, AR2534, AR2074, AR0533, AR1973, AR1613, AR1623, AR1633, AR2503, AR2213, AR1713, AR3113, AR2123, AR1653, AR2153, AR2172, AR3092, AR2632, AR1932, AR2822, AR1952, AR1682, AR1662, AR0892, AR1642, AR3122, AR3082, AR2232, AR1832, AR2672, AR1692, AR1982, AR2662, AR0332, AR2162, AR2712, AR2101, AR1921, AR2721, AR2401, AR2861, AR2771, AR0601, AR1801, AR2041, AR3131, AR2461, AR1041, AR2011, AR0961, AR2701, AR2051, AR2251, AR2471, AR2991, AR2581, AR1821, AR2741, AR2141, AR2871, AR2431 H0584: 3, H0341: 3, H0318: 2, H0271: 2, H0090: 2, H0264: 2, S0052: 2, H0521: 2, H0556: 1, H0740: 1, H0656: 1, H0580: 1, S0278: 1, H0431: 1, H0013: 1, H0284: 1, S0003: 1, S0144: 1, S0142: 1, S0002: 1, H0702: 1, L3832: 1, H0522: 1 and H0445: 1. 22 HPIBX03 743314 169 AR202: 71, AR194: 67, AR244: 58, AR281: 55, AR206: 54, AR241: 40, AR310: 38, AR315: 37, AR265: 36, AR283: 35, AR280: 35, AR246: 35, AR314: 34, AR104: 29, AR284: 29, AR186: 28, AR273: 26, AR292: 26, AR052: 25, AR198: 25, AR205: 25, AR243: 25, AR033: 23, AR039: 23, AR263: 22, AR271: 20, AR096: 20, AR282: 20, AR298: 20, AR251: 20, AR192: 19, AR204: 19, AR289: 18, AR259: 17, AR275: 17, AR274: 17, AR277: 17, AR055: 17, AR313: 16, AR291: 16, AR266: 16, AR286: 16, AR312: 15, AR285: 15, AR299: 15, AR218: 15, AR295: 15, AR240: 15, AR316: 14, AR309: 14, AR300: 14, AR247: 14, AR219: 14, AR294: 13, AR053: 13, AR213: 13, AR089: 12, AR061: 12, AR248: 12, AR249: 12, AR232: 12, AR177: 12, AR184: 12, AR296: 11, AR293: 11, AR268: 11, AR185: 11, AR270: 11, AR182: 10, AR227: 10, AR256: 10, AR290: 10, AR183: 10, AR269: 10, AR1759, AR2269, AR2589, AR2299, AR2389, AR2678, AR0608, AR2318, AR2378, AR1707, AR2337, AR2537, AR2347, AR2236, AR2256, AR1726, AR1715, AR1685, AR1765, AR2145, AR2175, AR2165, AR2245, AR2154, AR2214, AR1794, AR1614, AR1644, AR1664, AR2574, AR2224, AR2004, AR1623, AR1633, AR1803, AR2363, AR2613, AR1733, AR2553, AR1693, AR2723, AR2643, AR1653, AR2283, AR2883, AR2303, AR2873, AR1963, AR1813, AR2623, AR2392, AR1912, AR1932, AR1882, AR1902, AR1972, AR2102, AR1782, AR1952, AR1892, AR2122, AR2012, AR1741, AR2601, AR2971, AR2031 H0031: 4, S0442: 2, S0358: 2, S0150: 2, L0768: 2, S0152: 2, S0444: 1, S0132: 1, S0476: 1, H0620: 1, H0494: 1, S0002: 1, S0426: 1, L0776: 1, S0126: 1 and L0596: 1. 23 HDPBO81 892018 174 AR0533, AR2513, AR2623, AR2872, AR1642, AR2022, AR2522, AR1652, AR2822, AR1952, AR1682, AR1762, AR2612, AR2252, AR2572, AR2542, AR2972, AR2672, AR2942, AR2852, AR1722, AR2152, AR1622, AR2702, AR3092, AR2261, AR2141, AR2121, AR1931, AR2911, AR2131, AR1631, AR2171, AR2501, AR1751, AR0601, AR3121, AR2771, AR1611, AR1811, AR2341, AR1821, AR1961, AR1801, AR3001, AR0891, AR2211, AR2741 H0521: 6, L0804: 2, H0634: 1 and S0440: 1. 23 HDPBO81 790188 176 24 HWBFY57 837478 179 AR207: 12, AR245: 10, AR197: 10, AR250: 10, AR2019, AR2429, AR1959, AR2548, AR2528, AR2488, AR1847, AR1937, AR2127, AR0967, AR2646, AR1986, AR1666, AR2536, AR1656, AR1615, AR1625, AR1645, AR3085, AR2145, AR1635, AR2245, AR3115, AR2495, AR2465, AR2434, AR1924, AR2054, AR2714, AR2224, AR0534, AR2174, AR2234, AR2404, AR2724, AR2773, AR1743, AR2363, AR2043, AR2823, AR2633, AR2163, AR2133, AR1813, AR1823, AR1703, AR3123, AR2303, AR2883, AR1683, AR2703, AR2683, AR3163, AR2973, AR1833, AR1802, AR3092, AR2692, AR0602, AR3132, AR2852, AR0892, AR2962, AR2472, AR1782, AR1772, AR2292, AR2902, AR2952, AR0332, AR3002, AR2752, AR0522, AR2912, AR2892, AR2842, AR2662, AR0392, AR2342, AR2312, AR2612, AR2862, AR2982, AR2922, AR1732, AR1882, AR2872, AR2572, AR2992, AR0552, AR2062, AR2932, AR2282, AR1792, AR2111, AR2811, AR2621, AR2331, AR1851, AR2941, AR2261, AR2831, AR2321, AR2251, AR2351, AR2671, AR2741, AR2271, AR1861, AR1941, AR1751, AR0611, AR1891, AR1991, AR1041, AR2381, AR1711, AR2101 L0794: 6, H0580: 4, H0457: 3, L0785: 1, S0360: 1, S0002: 1, L0804: 1, L0806: 1, L0805: 1 and L0789: 1. 25 HYABV21 1281466 182 L0766: 6, L0748: 3, H0656: 2, H0264: 2, H0583: 1, H0650: 1, H0657: 1, L0655: 1, L0659: 1, L0790: 1, H0518: 1 and L0749: 1. 25 HYABV21 1213593 184 26 HOHBY69 827480 186 AR2445, AR2213, AR1683, AR2843, AR2823, AR1842, AR2242, AR1952, AR3102, AR1622, AR1612, AR1632, AR2641, AR1811, AR1821, AR1861, AR1711, AR2771, AR3081, AR2831, AR2631 S0250: 5, H0545: 2, S0210: 2, S0040: 1, S0342: 1, H0661: 1, S0418: 1, S0360: 1, H0549: 1, H0251: 1, H0544: 1, S0003: 1, H0124: 1, L0564: 1, L0807: 1, L0565: 1, S0037: 1, L0744: 1, L0757: 1 and S0276: 1. 26 HOHBY69 815681 188 27 HDHMA45 902513 202 AR2259, AR2778, AR2148, AR2238, AR2158, AR1657, AR1717, AR1646, AR1706, AR1686, AR1666, AR2246, AR2226, AR2356, AR1726, AR1625, AR2165, AR1615, AR2825, AR2175, AR2645, AR1635, AR2975, AR2215, AR2885, AR1805, AR3115, AR2074, AR2124, AR2614, AR2634, AR1784, AR2874, AR2574, AR2524, AR1833, AR1763, AR1923, AR0603, AR2913, AR3093, AR0893, AR2403, AR3083, AR2893, AR1813, AR1963, AR1733, AR2853, AR2833, AR2393, AR2623, AR2953, AR3163, AR2333, AR2963, AR2323, AR2003, AR2863, AR3123, AR1953, AR2283, AR2993, AR2133, AR2343, AR1913, AR2933, AR2383, AR2943, AR0963, AR1043, AR2473, AR2293, AR3003, AR1843, AR2663, AR2423, AR2713, AR2112, AR1692, AR2552, AR2452, AR2362, AR3132, AR2312, AR2582, AR2692, AR2012, AR2682, AR1982, AR2032, AR0392, AR2602, AR1792, AR1742, AR1902, AR2302, AR0552, AR1752, AR2902, AR2752, AR1852, AR0332, AR1772, AR1892, AR2702, AR2102, AR2052, AR2272, AR1882, AR2532, AR2432, AR2672, AR1822, AR2262, AR3102, AR2742, AR2022, AR2731, AR2721, AR1991, AR0531, AR2371, AR2541, AR2181, AR0611, AR2561, AR2651, AR2921, AR1931, AR2841 L0794: 5, L0769: 4, L0749: 3, S0110: 1, H0572: 1, H0050: 1, L0765: 1, L0756: 1, L0755: 1 and L0758: 1. 27 HDHMA45 812764 204 28 HMADJ14 1099342 206 AR184: 30, AR298: 28, AR259: 25, AR292: 24, AR249: 18, AR251: 17, AR229: 14, AR314: 13, AR237: 13, AR294: 13, AR296: 12, AR293: 12, AR233: 11, AR284: 11, AR227: 11, AR315: 11, AR182: 11, AR280: 10, AR266: 10, AR0399, AR2319, AR0619, AR0339, AR2488, AR2328, AR2868, AR2678, AR2818, AR2698, AR2268, AR2567, AR2897, AR2857, AR2347, AR2907, AR2387, AR1837, AR1777, AR2957, AR1756, AR0556, AR2916, AR1866, AR1856, AR1766, AR0895, AR2705, AR2685, AR2585, AR3005, AR1615, AR1625, AR1635, AR2285, AR2355, AR2635, AR2825, AR3135, AR3104, AR1794, AR0964, AR2194, AR1814, AR2014, AR1044, AR2184, AR3164, AR1724, AR0604, AR2774, AR2614, AR2994, AR2234, AR2023, AR1683, AR2833, AR0533, AR2453, AR2363, AR1653, AR2473, AR1643, AR2173, AR2553, AR3093, AR2713, AR2303, AR1663, AR2883, AR1913, AR2623, AR2403, AR1783, AR2533, AR2573, AR2523, AR2153, AR3113, AR2393, AR1963, AR1903, AR2873, AR2643, AR1713, AR2253, AR1733, AR2723, AR2002, AR2162, AR2742, AR2422, AR2972, AR2142, AR1932, AR1882, AR2032, AR2222, AR2242, AR2432, AR2752, AR2042, AR3122, AR1892, AR1992, AR2062, AR2072, AR1952, AR2652, AR2461, AR2111, AR3081, AR1741, AR1971, AR1801, AR2601, AR0521, AR2211 S0144: 6, S0278: 5, S0344: 3, S0002: 3, H0521: 3, S0003: 2, H0575: 1, S0214: 1, H0068: 1, H0591: 1, L0806: 1, L0805: 1, L0776: 1 and L0791: 1. 28 HMADJ14 889659 208 28 HMADJ14 843725 210 28 HMADJ14 843725 212 AR184:30, AR298:28, AR259:25, AR292:24, AR249:18, AR251:17, AR229:14, AR314:13, AR237:13, AR294:13, AR296:12, AR293:12, AR233:11, AR284:11, AR227:11, AR315:11, AR182:11, AR280:10, AR266:10, AR039:9, AR231:9, AR061:9, AR033:9, AR248:8, AR232:8, AR286:8, AR267:8, AR281:8, AR269:8, AR226:8, AR256:7, AR289:7, AR285:7, AR234:7, AR290:7, AR238:7, AR183:7, AR177:7, AR295:7, AR175:6, AR055:6, AR291:6, AR186:6, AR185:6, AR176:6, AR089:5, AR270:5, AR268:5, AR258:5, AR300:5, AR161:5, AR162:5, AR163:5, AR228:5, AR235:5, AR263:5, AR282:5, AR313:5, AR310:4, AR179:4, AR096:4, AR219:4, AR181:4, AR201:4, AR104:4, AR218:4, AR316:4, AR172:4, AR060:4, AR277:4, AR261:4, AR299:4, AR223:4, AR202:3, AR168:3, AR283:3, AR053:3, AR245:3, AR236:3, AR165:3, AR247:3, AR164:3, AR217:3, AR255:3, AR309:3, AR271:3, AR230:3, AR166:3, AR288:3, AR191:3, AR262:3, AR240:3, AR178:3, AR253:3, AR257:3, AR252:3, AR215:3, AR311:3, AR239:3, AR196:3, AR190:3, AR287:3, AR264:3, AR171:3, AR225:3, AR173:3, AR272:3, AR200:2, AR216:2, AR274:2, AR242:2, AR297:2, AR214:2, AR193:2, AR188:2, AR203:2, AR222:2, AR224:2, AR243:2, AR275:2, AR204:2, AR312:2, AR189:2, AR199:2, AR206:2, AR207:2, AR195:2, AR265:2, AR246:1, AR211:1, AR308:1, AR174:1, AR197:1, AR180:1, AR260:1, AR052:1, AR221:1, S0144:6, S0278:5, S0344:3, S0002:3, H0521:3, S0003:2, H0575:1, S0214:1, H0068:1, H0591:1, L0806:1, L0805:1, L0776:1 and L0791:1. 28 HMADJ14 795479 214 28 HMADJ14 426068 216 30 HEMFA84 608198 222 AR0606, AR0894, AR0554, AR2194, AR2823, AR3163, AR0963, AR1043, AR2183, AR2403, AR3132, AR2992, AR2472, AR2922, AR2832, AR0392, AR2462, AR2942, AR1852, AR3002, AR2481, AR2681, AR2771, AR3121, AR0331, AR1831, AR2631, AR2671, AR3101, AR0531, AR2381 L0747: 5, H0556: 4, L0769: 4, L0806: 3, L5622: 3, L0790: 3, L0779: 3, H0265: 2, H0013: 2, L0666: 2, L0439: 2, L0740: 2, L0749: 2, L0752: 2, L0757: 2, L0596: 2, T0049: 1, S0046: 1, S0132: 1, S0476: 1, L0717: 1, H0549: 1, H0600: 1, H0559: 1, H0046: 1, H0083: 1, H0266: 1, S0314: 1, H0622: 1, H0674: 1, H0135: 1, H0090: 1, L0564: 1, H0494: 1, S0440: 1, L0772: 1, L0800: 1, L0644: 1, L0794: 1, L0766: 1, L0776: 1, L0807: 1, L0809: 1, L0791: 1, L0665: 1, H0144: 1, H0660: 1, S0406: 1, L0780: 1, S0434: 1 and L0595: 1. 31 HDPPA04 904765 224 AR2517, AR1806, AR2524, AR1944, AR2494, AR1974, AR1693, AR1783, AR2353, AR2413, AR1903, AR1842, AR1722, AR2902, AR2712, AR1912, AR1742, AR2252, AR1662, AR2732, AR1752, AR2682, AR2242, AR2142, AR2912, AR1642, AR2822, AR1682, AR2532, AR1652, AR2952, AR2122, AR1832, AR2461, AR2871, AR0961, AR3111, AR2721, AR1881, AR3081, AR2041, AR2771, AR3101, AR2971, AR2011, AR2191, AR1861, AR2101, AR2131, AR2301, AR1891, AR2811, AR3131, AR2571, AR2021 H0521: 4, L0731: 4, H0591: 2, H0641: 2, L0794: 2, T0049: 1, S0476: 1, H0004: 1, H0494: 1, L0791: 1, H0522: 1, L0758: 1 and S0452: 1. 31 HDPPA04 905419 226 31 HDPPA04 905418 228 32 HE2OA95 637595 230 AR223:26, AR089:25, AR216:25, AR224:23, AR222:22, AR214:22, AR215:21, AR308:21, AR210:21, AR272:21, AR221:19, AR219:18, AR235:18, AR211:17, AR261:17, AR225:17, AR218:17, AR309:17, AR217:16, AR283:16, AR311:16, AR172:16, AR196:16, AR168:16, AR274:15, AR169:15, AR171:15, AR199:15, AR316:15, AR104:15, AR188:14, AR170:14, AR264:13, AR096:13, AR295:12, AR313:12, AR312:11, AR285:11, AR165:11, AR236:11, AR189:11, AR055:11, AR247:11, AR164:11, AR263:11, AR299:10, AR166:10, AR282:10, AR161:10, AR162:10, AR200:10, AR297:9, AR163:9, AR293:9, AR288:9, AR262:9, AR296:9, AR240:9, AR060:8, AR191:8, AR039:8, AR291:8, AR181:8, AR277:8, AR203:8, AR177:8, AR255:7, AR275:7, AR231:7, AR185:7, AR190:7, AR174:6, AR290:6, AR287:6, AR173:6, AR175:6, AR286:6, AR300:6, AR183:5, AR270:5, AR269:5, AR267:5, AR258:5, AR239:5, AR294:5, AR179:5, AR289:5, AR256:5, AR268:5, AR257:5, AR061:4, AR266:4, AR230:4, AR178:4, AR234:4, AR176:4, AR232:4, AR238:4, AR229:4, AR237:3, AR180:3, AR260:3, AR226:3, AR233:3, AR227:3, AR182:3, AR228:2, AR033:2, AR197:2, AR195:2, AR205:1, AR201:1, L0005:4, L0439:4, H0013:3, H0674:3, H0144:3, H0170:2, L0157:2, L0435:2, L0540:2, L0438:2, L0756:2, L0759:2, S0045:1, H0599:1, H0178:1, H0572:1, H0050:1, H0024:1, H0051:1, S6028:1, L0142:1, H0673:1, H0616:1, H0551:1, H0623:1, L0809:1, L0666:1, H0520:1, H0547:1, H0519:1, S0126:1, H0539:1, H0696:1, H0694:1, L0742:1, L0777:1, L0758:1, L0592:1 and L0595:1. 33 HKABZ65 862030 232 AR313: 41, AR242: 32, AR039: 28, AR165: 25, AR163: 25, AR164: 24, AR161: 24, AR162: 24, AR166: 24, AR089: 24, AR096: 23, AR173: 22, AR196: 20, AR193: 20, AR299: 20, AR300: 20, AR258: 20, AR180: 19, AR175: 19, AR178: 18, AR240: 18, AR229: 18, AR234: 18, AR185: 17, AR247: 17, AR218: 17, AR262: 17, AR179: 16, AR285: 16, AR183: 16, AR269: 16, AR293: 15, AR174: 15, AR199: 15, AR182: 15, AR181: 15, AR238: 14, AR191: 14, AR296: 14, AR236: 14, AR257: 14, AR316: 14, AR270: 14, AR226: 13, AR219: 13, AR297: 13, AR277: 13, AR264: 12, AR200: 12, AR312: 12, AR195: 12, AR213: 12, AR192: 12, AR203: 12, AR268: 12, AR212: 12, AR294: 12, AR286: 11, AR060: 11, AR230: 11, AR177: 11, AR189: 11, AR233: 11, AR260: 10, AR231: 10, AR198: 10, AR290: 10, AR188: 10, AR204: 10, AR0539, AR2879, AR2889, AR2559, AR2959, AR0339, AR2619, AR2829, AR1049, AR2459, AR2439, AR2359, AR2288, AR3088, AR2638, AR2758, AR2918, AR2018, AR2748, AR2377, AR1977, AR2397, AR2247, AR3117, AR1767, AR2677, AR1727, AR2567, AR2237, AR2057, AR1716, AR2276, AR1686, AR2146, AR2076, AR1696, AR2256, AR2526, AR2506, AR2716, AR2156, AR1706, AR2116, AR2216, AR3095, AR2835, AR2665, AR2545, AR2225, AR1905, AR2105, AR2165, AR2175, AR2325, AR0555, AR2894, AR2534, AR2464, AR2723, AR0612 H0494: 1 33 HKABZ65 665424 234

Table 1C summarizes additional polynucleotides encompassed by the invention (including cDNA clones related to the sequences (Clone ID:), contig sequences (contig identifier (Contig ID:) contig nucleotide sequence identifiers (SEQ ID NO:X)), and genomic sequences (SEQ ID NO:B). The first column provides a unique clone identifier, “cDNA Clone ID”, for a cDNA clone related to each contig sequence. The second column provides the sequence identifier, “SEQ ID NO:X”, for each contig sequence. The third column provides a unique contig identifier, “Contig ID:” for each contig sequence. The fourth column, provides a BAC identifier “BAC ID: A” for the BAC clone referenced in the corresponding row of the table. The fifth column provides the nucleotide sequence identifier, “SEQ ID NO:B” for a fragment of the BAC clone identified in column four of the corresponding row of the table. The sixth column, “Exon From-To”, provides the location (i.e., nucleotide position numbers) within the polynucleotide sequence of SEQ ID NO:B which delineate certain polynucleotides of the invention that are also exemplary members of polynucleotide sequences that encode polypeptides of the invention (e.g., polypeptides containing amino acid sequences encoded by the polynucleotide sequences delineated in column six, and fragments and variants thereof). Table 1C from U.S. patent application Ser. No. 10/472,532, filed Sep. 20, 2003, is herein incorporated by reference. Table 1C in priority Application No. PCT/US02/09785, filed Mar. 19, 2002, which corresponds to Publication No. WO02/95010, published Nov. 28, 2002 (e.g., pages 228 to 235 of Publication No. WO02/95010) is incorporated by reference herein in its entirety.

TABLE 1C cDNA Clone SEQ ID CONTIG SEQ ID EXON ID NO: X ID: BAC ID: A NO: B From-To HETBX14 61 806447 AC011473 247  1-205  727-1286 1995-2071 2865-3025 3352-3617 4434-4573 4958-5431 HETBX14 61 806447 AC011473 248  1-77 241-299 745-906 1417-1520 2346-2501 2754-2852 2923-3002 3962-4096 5422-5791 HLHFP03 76 460467 AC011976 251  1-467 1023-1116 2101-2317 2529-2630 2860-3097 HLHFP03 76 460467 AC011976 252  1-391 HTADX17 90 457172 AL357565 249  1-937  945-1293 2068-2336 2732-3109 HTADX17 90 457172 AL357565 250  1-104 HJACG02 102 509948 AC008763  1-47 243-371 736-823 1144-1381 HJACG02 102 509948 AC008763  1-32 407-497  818-1357 2275-2380 2384-2560 HT5GJ57 159 740767 AC005081 253  1-149 1029-1450 4881-5071 5302-5397 6004-6132 6888-6926 7229-7329 9066-9254 9805-9848  9852-10211 10276-10315 10619-10807 10855-11472 11695-11852 12358-12468 13770-13847 14053-14259 14695-14822 16101-16553 18233-18690 18750-19965 HT5GJ57 159 740767 AC016675 254  1-149 1029-1450 1623-1790 4908-5098 5329-5424 6031-6159 6915-6953 7256-7356 9095-9306 9834-9877  9881-10239 10304-10343 10647-10835 10883-11500 11724-11881 13799-13876 14082-14288 14725-14852 16131-16583 18262-18719 18757-19994 20161-20420 HT5GJ57 159 740767 AC005081 255  1-233 HT5GJ57 159 740767 AC016675 256  1-720 2099-2265 2776-3550 3946-4146 4976-5104 5781-6171 6476-7039 7384-7738 7837-8009 8434-8593 11644-11746

Tables 1D, 1E, 1E.1, and 1E.2: The polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists could be used to treat the associated disease.

The present invention encompasses methods of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of treating a disease or disorder (such as a disease listed in the “Preferred Indications” columns of Tables 1D, 1E, 1E.2, or 1F, and in particular, an immune, cardiovascular, cancer, or other proliferative disease or disorder) comprising administering to a patient in which such detection, treatment, prevention, and/or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate the disease or disorder. The first and second columns of Table 1D show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in preventing, treating, diagnosing, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in Column 3 of Table 1D.

In another embodiment, the present invention also encompasses methods of detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating a disease or disorder (such as a disease listed in the “Preferred Indications” columns of Tables 1D, 1E, 1E.2, or 1F, and in particular, an immune, cardiovascular, cancer, or other proliferative disease or disorder); comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in Column 3 of Table 1D or in the “Preferred Indications” columns of Tables 1E, 1E.2, or 1F.

The “Preferred Indications” columns of Tables 1D, 1E, 1E.2, and 1F describe diseases, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).

The recitation of “Cancer” in the “Preferred Indications” columns indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., leukemias, cancers, and/or as described below under “Hyperproliferative Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D may be used for example, to diagnose, treat, prevent, and/or ameliorate a neoplasm located in a tissue selected from the group consisting of: colon, abdomen, bone, breast, digestive system, liver, pancreas, prostate, peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a pre-neoplastic condition, selected from the group consisting of: hyperplasia (e.g., endometrial hyperplasia and/or as described in the section entitled “Hyperproliferative Disorders”), metaplasia (e.g., connective tissue metaplasia, atypical metaplasia, and/or as described in the section entitled “Hyperproliferative Disorders”), and/or dysplasia (e.g., cervical dysplasia, and bronchopulmonary dysplasia).

In another specific embodiment, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a benign dysproliferative disorder selected from the group consisting of: benign tumors, fibrocystic conditions, tissue hypertrophy, and/or as described in the section entitled “Hyperproliferative Disorders”.

The recitation of “Immune/Hematopoietic” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having the “Immune/Hematopoietic” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: anemia, pancytopenia, leukopenia, thrombocytopenia, leukemias, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis, granulomatous disease, immune deficiency, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, systemic lupus erythematosis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and allergies.

The recitation of “Reproductive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the reproductive system (e.g., as described below under “Reproductive System Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Reproductive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cryptorchism, prostatitis, inguinal hernia, varicocele, leydig cell tumors, verrucous carcinoma, prostatitis, malacoplakia, Peyronie's disease, penile carcinoma, squamous cell hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner's syndrome, mucopurulent cervicitis, Sertoli-leydig tumors, ovarian cancer, uterine cancer, pelvic inflammatory disease, testicular cancer, prostate cancer, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, testicular atrophy, testicular feminization, anorchia, ectopic testis, epididymitis, orchitis, gonorrhea, syphilis, testicular torsion, vasitis nodosa, germ cell tumors, stromal tumors, dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding, cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, cervical neoplasms, pseudohermaphroditism, and premenstrual syndrome.

The recitation of “Musculoskeletal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the immune system (e.g., as described below under “Immune Activity”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Musculoskeletal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bone cancers (e.g., osteochondromas, benign chondromas, chondroblastoma, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, multiple myeloma, osteosarcomas), Paget's Disease, rheumatoid arthritis, systemic lupus erythematosus, osteomyelitis, Lyme Disease, gout, bursitis, tendonitis, osteoporosis, osteoarthritis, muscular dystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis.

The recitation of “Cardiovascular” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cardiovascular” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: myxomas, fibromas, rhabdomyomas, cardiovascular abnormalities (e.g., congenital heart defects, cerebral arteriovenous malformations, septal defects), heart disease (e.g., heart failure, congestive heart disease, arrhythmia, tachycardia, fibrillation, pericardial Disease, endocarditis), cardiac arrest, heart valve disease (e.g., stenosis, regurgitation, prolapse), vascular disease (e.g., hypertension, coronary artery disease, angina, aneurysm, arteriosclerosis, peripheral vascular disease), hyponatremia, hypernatremia, hypokalemia, and hyperkalemia.

The recitation of “Mixed Fetal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Mixed Fetal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: spina bifida, hydranencephaly, neurofibromatosis, fetal alcohol syndrome, diabetes mellitus, PKU, Down's syndrome, Patau syndrome, Edwards syndrome, Turner syndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crouzon syndrome, cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel-Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome, Smith-Lemli-Opitz syndrome, thromocytopenia-absent radius (TAR) syndrome, Treacher Collins syndrome, Williams syndrome, Hirschsprung's disease, Meckel's diverticulum, polycystic kidney disease, Turner's syndrome, and gonadal dysgenesis, Klippel-Feil syndrome, Ostogenesis imperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumor, neuroblastoma, and retinoblastoma.

The recitation of “Excretory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and renal disorders (e.g., as described below under “Renal Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Excretory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bladder cancer, prostate cancer, benign prostatic hyperplasia, bladder disorders (e.g., urinary incontinence, urinary retention, urinary obstruction, urinary tract Infections, interstitial cystitis, prostatitis, neurogenic bladder, hematuria), renal disorders (e.g., hydronephrosis, proteinuria, renal failure, pyelonephritis, urolithiasis, reflux nephropathy, and unilateral obstructive uropathy).

The recitation of “Neural/Sensory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the nervous system (e.g., as described below under “Neural Activity and Neurological Diseases”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Neural/Sensory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: brain cancer (e.g., brain stem glioma, brain tumors, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, and cerebral astrocytoma, neurodegenerative disorders (e.g., Alzheimer's Disease, Creutzfeldt-Jakob Disease, Parkinson's Disease, and Idiopathic Presenile Dementia), encephalomyelitis, cerebral malaria, meningitis, metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylase deficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS Dementia Complex, schizophrenia, attention deficit disorder, hyperactive attention deficit disorder, autism, and obsessive compulsive disorders.

The recitation of “Respiratory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Respiratory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of the respiratory system such as larynx cancer, pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamous cell carcinomas, small cell (oat cell) carcinomas, large cell carcinomas, and adenocarcinomas. Allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X, infiltrative lung diseases (e.g., pulmonary fibrosis and lymphoid interstitial pneumonia), obstructive airway diseases (e.g., asthma, emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis and asbestosis), pneumonia, and pleurisy.

The recitation of “Endocrine” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”), renal disorders (e.g., as described below under “Renal Disorders”), and disorders of the endocrine system (e.g., as described below under “Endocrine Disorders”.

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having an “Endocrine” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of endocrine tissues and organs (e.g., cancers of the hypothalamus, pituitary gland, thyroid gland, parathyroid glands, pancreas, adrenal glands, ovaries, and testes), diabetes (e.g., diabetes insipidus, type I and type II diabetes mellitus), obesity, disorders related to pituitary glands (e.g., hyperpituitarism, hypopituitarism, and pituitary dwarfism), hypothyroidism, hyperthyroidism, goiter, reproductive disorders (e.g. male and female infertility), disorders related to adrenal glands (e.g., Addison's Disease, corticosteroid deficiency, and Cushing's Syndrome), kidney cancer (e.g., hypernephroma, transitional cell cancer, and Wilm's tumor), diabetic nephropathy, interstitial nephritis, polycystic kidney disease, glomerulonephritis (e.g., IgM mesangial proliferative glomerulonephritis and glomerulonephritis caused by autoimmune disorders; such as Goodpasture's syndrome), and nephrocalcinosis.

The recitation of “Digestive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the gastrointestinal system (e.g., as described below under “Gastrointestinal Disorders”.

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Digestive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portal hypertension, cholelithiasis, cancer of the digestive system (e.g., biliary tract cancer, stomach cancer, colon cancer, gastric cancer, pancreatic cancer, cancer of the bile duct, tumors of the colon (e.g., polyps or cancers), and cirrhosis), pancreatitis, ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atropy, benign tumors of the duodenum, distension, irritable bowel syndrome, malabsorption, congenital disorders of the small intestine, bacterial and parasitic infection, megacolon, Hirschsprung's disease, aganglionic megacolon, acquired megacolon, colitis, anorectal disorders (e.g., anal fistulas, hemorrhoids), congenital disorders of the liver (e.g., Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and alpha1-antitrypsin deficiency), portal hypertension, cholelithiasis, and jaundice.

The recitation of “Connective/Epithelial” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), and or to promote or inhibit regeneration (e.g., as described below under “Regeneration”), and wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Connective/Epithelial” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: connective tissue metaplasia, mixed connective tissue disease, focal epithelial hyperplasia, epithelial metaplasia, mucoepithelial dysplasia, graft v. host disease, polymyositis, cystic hyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer's disease, lymphoproliferative disorder, Waldenstron's macroglobulinemia, Crohn's disease, pernicious anemia, idiopathic Addison's disease, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease, wound healing, relapsing polychondritis, vasculitis, polyarteritis nodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderma, CREST syndrome, Sjogren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease, relapsing polychondritis, vasculitis, Henoch-Schonlein syndrome, erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis, Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, cellulitis, keloids, Ehler Danlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogenese imperfecta, chondrodysplasias, epidermolysis bullosa, Alport syndrome, and cutis laxa.

TABLE 1D Gene No. cDNA Clone ID Preferred Indications 3 HTEEB42 Cancer 4 HEMCM42 Cancer 4 HEQCC55 Cancer 5 HEMAE80 Cardiovascular, Musculoskeletal, Reproductive 6 HRDFB85 Cancer 7 HDTAW95 Cancer 8 HEMCV19 Cancer 9 HETBX14 Cancer 10 HLHSK94 Cancer 11 HLHFP03 Respiratory 11 HLHFP03 Respiratory 12 HHTLF25 Cancer 13 HTADX17 Immune/Hematopoetic, Reproductive 13 HTADX17 Immune/Hematopoetic, Reproductive 14 HJACG02 Digestive, Immune/Hematopoetic 15 HKGAJ54 Cancer 16 HE8CH92 Cancer 16 HSVAK93 Cancer 17 HSDEK49 Cancer 18 HWBAO62 Connective/Epithelial, Immune/Hematopoetic 19 HWHGU54 Connective/Epithelial 20 HCEJQ69 Cancer 21 HT5GJ57 Cancer 21 HT5GJ57 Cancer 22 HPIBX03 Cancer 23 HDPBO81 Digestive, Immune/Hematopoetic, Reproductive 24 HWBFY57 Digestive, Immune/Hematopoetic 25 HYABV21 Immune/Hematopoietic 26 HOHBY69 Cancer 27 HDHMA45 Cardiovascular, Neural/Sensory 28 HMADJ14 Connective/Epithelial, Immune/Hematopoetic, Musculoskeletal 28 HMADJ14 Connective/Epithelial, Immune/Hematopoietic, Musculoskeletal 30 HEMFA84 Cancer 31 HDPPA04 Cardiovascular, Connective/Epithelial, Immune/Hematopoetic 32 HE2OA95 Cancer 33 HKABZ65 Connective/Epithelial

Table 1E provides information related to biological activities and preferred indications for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1E also provides information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“cDNA Clone ID”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B.1, 1B.2, 1C, and 1D. The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A and 1B.1). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The sixth column (“Preferred Indications”) describes particular embodiments of the invention as well as indications (e.g. pathologies, diseases, disorders, abnormalities, etc.) for which polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) may be used in detecting, diagnosing, preventing, and/or treating.

TABLE 1E AA SEQ Gene cDNA ID Biological No. Clone ID NO: Y Activity Exemplary Activity Assay Preferred Indication 3 HTEEB42 10 Regulation of Assays for the regulation of A highly preferred indication is transcription of transcription of Malic Enzyme ‘diabetes mellitus. An additional highly Malic Enzyme are well-known in the art and preferred indication is a complication in hepatocytes may be used or routinely associated with diabetes (e.g., diabetic modified to assess the ability of retinopathy, diabetic nephropathy, kidney polypeptides of the invention disease (e.g., renal failure, nephropathy (including antibodies and and/or other diseases and disorders as agonists or antagonists of the described in the “Renal Disorders” section invention) to regulate below), diabetic neuropathy, nerve disease transcription of Malic Enzyme, and nerve damage (e.g., due to diabetic a key enzyme in lipogenesis. neuropathy), blood vessel blockage, heart Malic enzyme is involved in disease, stroke, impotence (e.g., due to lipogenesisand its expression is diabetic neuropathy or blood vessel stimulted by insulin. ME blockage), seizures, mental confusion, promoter contains two direct drowsiness, nonketotic hyperglycemic- repeat (DR1)-like elements hyperosmolar coma, cardiovascular disease MEp and MEd identified as (e.g., heart disease, atherosclerosis, putative PPAR response microvascular disease, hypertension, elements. ME promoter may stroke, and other diseases and disorders as also responds to AP1 and other described in the “Cardiovascular Disorders” transcription factors. section below), dyslipidemia, endocrine Exemplary assays that may be disorders (as described in the “Endocrine used or routinely modified to Disorders” section below), neuropathy, test for regulation of vision impairment (e.g., diabetic transcription of Malic Enzyme retinopathy and blindness), ulcers and (in hepatocytes) by impaired wound healing, and infection polypeptides of the invention (e.g., infectious diseases and disorders as (including antibodies and described in the “Infectious Diseases” agonists or antagonists of the section below, especially of the urinary invention) include assays tract and skin), carpal tunnel syndrome and disclosed in: Streeper, R. S., et Dupuytren's contracture). An al., Mol Endocrinol, additional highly preferred indication is 12(11): 1778-91 (1998); Garcia- obesity and/or complications associated Jimenez, C., et al., Mol with obesity. Additional highly preferred Endocrinol, 8(10): 1361-9 indications include weight loss or (1994); Barroso, I., et al., J Biol alternatively, weight gain. Chem, 274(25): 17997-8004 Aditional highly preferred indications are (1999); Ijpenberg, A., et al., J complications associated with insulin Biol Chem, 272(32): 20108-20117 resistance. (1997); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays are publicly available (e.g., through the ATCC ™) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays includes the mouse 3T3-L1 cell line. 3T3-L1 is a mouse preadipocyte cell line (adherent). It is a continuous substrain of 3T3 fibroblasts developed through clonal isolation. Cells undergo a pre- adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 4 HEQCC55 16 Production of MCP-1 FMAT. Assays for A highly preferred embodiment of the MCP-1 immunomodulatory proteins invention includes a method for stimulating that are produced by a large (e.g., increasing) MCP-1 production. An variety of cells and act to alternative highly preferred embodiment of induce chemotaxis and the invention includes a method for activation of monocytes and T inhibiting (e.g., reducing) MCP-1 cells are well known in the art production. A highly preferred indication and may be used or routinely is infection (e.g., an infectious disease as modified to assess the ability of described below under “Infectious polypeptides of the invention Disease”). Additional highly preferred (including antibodies and indications include inflammation and agonists or antagonists of the inflammatory disorders. Preferred invention) to mediate indications include blood disorders (e.g., as immunomodulation, induce described below under “Immune Activity”, chemotaxis, and modulate “Blood-Related Disorders”, and/or immune cell activation. “Cardiovascular Disorders”). Highly Exemplary assays that test for preferred indications include autoimmune immunomodulatory proteins diseases (e.g., rheumatoid arthritis, evaluate the production of cell systemic lupus erythematosis, multiple surface markers, such as sclerosis and/or as described below) and monocyte chemoattractant immunodeficiencies (e.g., as described protein (MCP), and the below). Preferred indications also activation of monocytes and T include anemia, pancytopenia, leukopenia, cells. Such assays that may be thrombocytopenia, Hodgkin's disease, used or routinely modified to acute lymphocytic anemia (ALL), test immunomodulatory and plasmacytomas, multiple myeloma, diffferentiation activity of Burkitt's lymphoma, arthritis, AIDS, polypeptides of the invention granulomatous disease, inflammatory (including antibodies and bowel disease, sepsis, neutropenia, agonists or antagonists of the neutrophilia, psoriasis, suppression of invention) include assays immune reactions to transplanted organs disclosed in Miraglia et al., J and tissues, hemophilia, hypercoagulation, Biomolecular Screening 4: 193-204 diabetes mellitus, endocarditis, meningitis (1999); Rowland et al., (bacterial and viral), Lyme Disease, “Lymphocytes: a practical asthma, and allergy Preferred indications approach” Chapter 6: 138-160 also include neoplastic diseases (e.g., (2000); Satthaporn and Eremin, leukemia, lymphoma, and/or as described J R Coll Surg Ednb 45(1): 9-19 below under “Hyperproliferative (2001); and Verhasselt et al., J Disorders”). Highly preferred indications Immunol 158: 2919-2925 include neoplasms and cancers, such as, (1997), the contents of each of leukemia, lymphoma, prostate, breast, lung, which are herein incorporated colon, pancreatic, esophageal, stomach, by reference in its entirety. brain, liver, and urinary cancer. Other Human dendritic cells that may preferred indications include benign be used according to these dysproliferative disorders and pre- assays may be isolated using neoplastic conditions, such as, for example, techniques disclosed herein or hyperplasia, metaplasia, and/or dysplasia. otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 4 HEQCC55 16 Production of Assays for production of IL-13 Highly preferred indications include allergy IL-13 and and activation of T-cells are and asthma. Additional highly preferred activation of T- well known in the art and may indications include immune and cells. be used or routinely modified to hematopoietic disorders (e.g., as described assess the ability of below under “Immune Activity”, and polypeptides of the invention “Blood-Related Disorders”), autoimmune (including antibodies and diseases (e.g., rheumatoid arthritis, agonists or antagonists of the systemic lupus erythematosis, Crohn″s invention) to stimulate or disease, multiple sclerosis and/or as inhibit production of IL-13 described below), immunodeficiencies and/or activation of T-cells. (e.g., as described below), boosting a T Exemplary assays for IL-13 cell-mediated immune response, and production that may be used or suppressing a T cell-mediated immune routinely modified to test response. activity of polypeptides and antibodies of the invention (including agonists or antagonists of the invention) include, for example, assays such as disclosed and/or cited in: Grunig, G, et al., “Requirement for IL-13 independently of IL-4 in Experimental asthma” Science; 282: 2261-2263 (1998), and Wills-Karp M, et al., “Interleukin-13: central mediator of allergic asthma” Science; 282: 2258-2261 (1998); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL13, a Th2 type cytokine, is a potent stimulus for mucus production, airway hyper-responsiveness and allergic asthma. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated in in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 11 HLHFP03 77 Activation of Kinase assay. JNK and p38 Preferred indications include neoplastic T-Cell p38 or kinase assays for signal diseases (e.g., as described below under JNK Signaling transduction that regulate cell “Hyperproliferative Disorders”), blood Pathway. proliferation, activation, or disorders (e.g., as described below under apoptosis are well known in the “Immune Activity”, “Cardiovascular art and may be used or Disorders”, and/or “Blood-Related routinely modified to assess the Disorders”), and infection (e.g., an ability of polypeptides of the infectious disease as described below under invention (including antibodies “Infectious Disease”). Highly preferred and agonists or antagonists of indications include autoimmune diseases the invention) to promote or (e.g., rheumatoid arthritis, systemic lupus inhibit immune cell (e.g. T-cell) erythematosis, multiple sclerosis and/or as proliferation, activation, and described below) and immunodeficiencies apoptosis. Exemplary assays (e.g., as described below). Additional for JNK and p38 kinase activity highly preferred indications include that may be used or routinely inflammation and inflammatory disorders. modified to test JNK and p38 Highly preferred indications also include kinase-induced activity of neoplastic diseases (e.g., leukemia, polypeptides of the invention lymphoma, and/or as described below (including antibodies and under “Hyperproliferative Disorders”). agonists or antagonists of the Highly preferred indications include invention) include the assays neoplasms and cancers, such as, leukemia, disclosed in Forrer et al., Biol lymphoma, prostate, breast, lung, colon, Chem 379(8-9): 1101-1110 pancreatic, esophageal, stomach, brain, (1998); Gupta et al., Exp Cell liver, and urinary cancer. Other preferred Res 247(2): 495-504 (1999); indications include benign dysproliferative Kyriakis JM, Biochem Soc disorders and pre-neoplastic conditions, Symp 64: 29-48 (1999); Chang such as, for example, hyperplasia, and Karin, Nature metaplasia, and/or dysplasia. Preferred 410(6824): 37-40 (2001); and indications include arthritis, asthma, AIDS, Cobb MH, Prog Biophys Mol allergy, anemia, pancytopenia, leukopenia, Biol 71(3-4): 479-500 (1999); thrombocytopenia, Hodgkin″s disease, the contents of each of which acute lymphocytic anemia (ALL), are herein incorporated by plasmacytomas, multiple myeloma, reference in its entirety. T cells Burkitt″s lymphoma, granulomatous that may be used according to disease, inflammatory bowel disease, these assays are publicly sepsis, psoriasis, suppression of immune available (e.g., through the reactions to transplanted organs and tissues, ATCC ™). Exemplary mouse T endocarditis, meningitis, and Lyme cells that may be used Disease. according to these assays include the CTLL cell line, which is an IL-2 dependent suspension-culture cell line with cytotoxic activity. 11 HLHFP03 79 Activation of Kinase assay. JNK and p38 Preferred indications include neoplastic T-Cell p38 or kinase assays for signal diseases (e.g., as described below under JNK Signaling transduction that regulate cell “Hyperproliferative Disorders”), blood Pathway. proliferation, activation, or disorders (e.g., as described below under apoptosis are well known in the “Immune Activity”, “Cardiovascular art and may be used or Disorders”, and/or “Blood-Related routinely modified to assess the Disorders”), and infection (e.g., an ability of polypeptides of the infectious disease as described below under invention (including antibodies “Infectious Disease”). Highly preferred and agonists or antagonists of indications include autoimmune diseases the invention) to promote or (e.g., rheumatoid arthritis, systemic lupus inhibit immune cell (e.g. T-cell) erythematosis, multiple sclerosis and/or as proliferation, activation, and described below) and immunodeficiencies apoptosis. Exemplary assays (e.g., as described below). Additional for JNK and p38 kinase activity highly preferred indications include that may be used or routinely inflammation and inflammatory disorders. modified to test JNK and p38 Highly preferred indications also include kinase-induced activity of neoplastic diseases (e.g., leukemia, polypeptides of the invention lymphoma, and/or as described below (including antibodies and under “Hyperproliferative Disorders”). agonists or antagonists of the Highly preferred indications include invention) include the assays neoplasms and cancers, such as, leukemia, disclosed in Forrer et al., Biol lymphoma, prostate, breast, lung, colon, Chem 379(8-9): 1101-1110 pancreatic, esophageal, stomach, brain, (1998); Gupta et al., Exp Cell liver, and urinary cancer. Other preferred Res 247(2): 495-504 (1999); indications include benign dysproliferative Kyriakis JM, Biochem Soc disorders and pre-neoplastic conditions, Symp 64: 29-48 (1999); Chang such as, for example, hyperplasia, and Karin, Nature metaplasia, and/or dysplasia. Preferred 410(6824): 37-40 (2001); and indications include arthritis, asthma, AIDS, Cobb MH, Prog Biophys Mol allergy, anemia, pancytopenia, leukopenia, Biol 71(3-4): 479-500 (1999); thrombocytopenia, Hodgkin″s disease, the contents of each of which acute lymphocytic anemia (ALL), are herein incorporated by plasmacytomas, multiple myeloma, reference in its entirety. T cells Burkitt″s lymphoma, granulomatous that may be used according to disease, inflammatory bowel disease, these assays are publicly sepsis, psoriasis, suppression of immune available (e.g., through the reactions to transplanted organs and tissues, ATCC ™). Exemplary mouse T endocarditis, meningitis, and Lyme cells that may be used Disease. according to these assays include the CTLL cell line, which is an IL-2 dependent suspension-culture cell line with cytotoxic activity. 11 HLHFP03 79 SEAP in HIB/CRE 11 HLHFP03 79 VEGF in HT1080 11 HLHFP03 79 Production of Assays for measuring Highly preferred indications include VCAM in expression of VCAM are well- inflammation (acute and chronic), endothelial known in the art and may be restnosis, atherosclerosis, asthma and cells (such as used or routinely modified to allergy. Highly preferred indications vessels; thus VCAM expression plays a role in promoting immune and inflammatory responses. 11 HLHFP03 79 SEAP in OE- 21 12 HHTLF25 84 Production of IL-10 FMAT. Assays for A highly preferred embodiment of the IL-10 and immunomodulatory proteins invention includes a method for stimulating downregulation produced by activated T cells, the production of IL-10. An alternative of immune B cells, and monocytes that preferred embodiment of the invention responses exhibit anti-inflammatory includes a method for inhibiting the activity and downregulate production of IL-10. Highly preferred monocyte/macrophage function indications include inflammation and and expression of cytokines are inflammatory disorders (e.g. inflammatory well known in the art and may bowel disease). An additional highly be used or routinely modified to preferred indication includes inflammatory assess the ability of the bowel disease. Additional highly polypeptides of the invention preferred indications include blood (including antibodies and disorders (e.g., as described below under agonists or antagonists of the “Immune Activity” (e.g. autoimmune invention) to mediate disorders), “Blood-Related Disorders”, immunomodulation, regulate and/or “Cardiovascular Disorders”). Highly inflammatory activities, and preferred indications include autoimmune modulate immune cell function diseases (e.g., rheumatoid arthritis, and cytokine production. systemic lupus erythematosis, multiple Exemplary assays that test for sclerosis and/or as described below) and immunomodulatory proteins immunodeficiencies (e.g., as described evaluate the production of below). Preferred indications include cytokines, such as IL-10, and neoplastic diseases (e.g., leukemia, the downmodulation of immune lymphoma, melanoma, and/or as described responses. Such assays that below under “Hyperproliferative may be used or routinely Disorders”). Preferred indications include modified to test neoplasms and cancers, such as, for immunomodulatory activity of example, leukemia, lymphoma, melanoma, polypeptides of the invention and prostate, breast, lung, colon, (including antibodies and pancreatic, esophageal, stomach, brain, agonists or antagonists of the liver and urinary cancer. Other preferred invention) include the assays indications include benign dysproliferative disclosed in Miraglia et al., J disorders and pre-neoplastic conditions, Biomolecular Screening 4: 193-204 such as, for example, hyperplasia, (1999); Rowland et al., metaplasia, and/or dysplasia. Preferred “Lymphocytes: a practical indications include anemia, pancytopenia, approach” Chapter 6: 138-160 leukopenia, thrombocytopenia, Hodgkin's (2000); and Koning et al., disease, acute lymphocytic anemia (ALL), Cytokine 9(6): 427-436 (1997), plasmacytomas, multiple myeloma, the contents of each of which Burkitt's lymphoma, Crohn″s disease, are herein incorporated by arthritis, AIDS, granulomatous disease, reference in its entirety. sepsis, neutropenia, neutrophilia, psoriasis, Human T cells that may be used suppression of immune reactions to according to these assays may transplanted organs and tissues, be isolated using techniques hemophilia, hypercoagulation, diabetes disclosed herein or otherwise mellitus, endocarditis, meningitis, Lyme known in the art. Human T Disease, asthma and allergy. An cells are primary human additional preferred indication is infection lymphocytes that mature in the (e.g., as described below under “Infectious thymus and express a T cell Disease”). receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 13 HTADX17 91 Activation of Assays for the activation of Highly preferred indications include transcription transcription through the blood disorders (e.g., as described below through NFAT Nuclear Factor of Activated T under “Immune Activity”, “Blood-Related response in cells (NFAT) response element Disorders”, and/or ““Cardiovascular immune cells are well-known in the art and Disorders””). Highly preferred indications (such as T- may be used or routinely include autoimmune diseases (e.g., cells). modified to assess the ability of rheumatoid arthritis, systemic lupus polypeptides of the invention erythematosis, multiple sclerosis and/or as (including antibodies and described below), immunodeficiencies agonists or antagonists of the (e.g., as described below), boosting a T invention) to regulate NFAT cell-mediated immune response, and transcription factors and suppressing a T cell-mediated immune modulate expression of genes response. Additional highly preferred involved in immunomodulatory indications include inflammation and functions. Exemplary assays inflammatory disorders. An additional for transcription through the highly preferred indication is infection NFAT response element that (e.g., an infectious disease as described may be used or routinely below under “Infectious Disease”). modified to test NFAT- Preferred indications include neoplastic response element activity of diseases (e.g., leukemia, lymphoma, and/or polypeptides of the invention as described below under (including antibodies and “Hyperproliferative Disorders”). Preferred agonists or antagonists of the indications include neoplasms and cancers, invention) include assays such as, for example, leukemia, lymphoma, disclosed in Berger et al., Gene and prostate, breast, lung, colon, 66: 1-10 (1998); Cullen and pancreatic, esophageal, stomach, brain, Malm, Methods in Enzymol liver and urinary cancer. Other preferred 216: 362-368 (1992); Henthorn indications include benign dysproliferative et al., Proc Natl Acad Sci USA disorders and pre-neoplastic conditions, 85: 6342-6346 (1988); Serfling such as, for example, hyperplasia, et al., Biochim Biophys Acta metaplasia, and/or dysplasia. Preferred 1498(1): 1-18 (2000); De Boer indications also include anemia, et al., Int J Biochem Cell Biol pancytopenia, leukopenia, 31(10): 1221-1236 (1999); thrombocytopenia, Hodgkin's disease, Fraser et al., Eur J Immunol acute lymphocytic anemia (ALL), 29(3): 838-844 (1999); and plasmacytomas, multiple myeloma, Yeseen et al., J Biol Chem Burkitt's lymphoma, arthritis, AIDS, 268(19): 14285-14293 (1993), granulomatous disease, inflammatory the contents of each of which bowel disease, sepsis, neutropenia, are herein incorporated by neutrophilia, psoriasis, suppression of reference in its entirety. T cells immune reactions to transplanted organs that may be used according to and tissues, hemophilia, hypercoagulation, these assays are publicly diabetes mellitus, endocarditis, meningitis, available (e.g., through the Lyme Disease, asthma and allergy. ATCC ™). Exemplary human T cells that may be used according to these assays include the JURKAT cell line, which is a suspension culture of leukemia cells that produce IL- 2 when stimulated. 13 HTADX17 91 Activation of Assays for the activation of Highly preferred indications include transcription transcription through the neoplastic diseases (e.g., leukemia, through GAS Gamma Interferon Activation lymphoma, and/or as described below response Site (GAS) response element under “Hyperproliferative Disorders”). element in are well-known in the art and Highly preferred indications include immune cells may be used or routinely neoplasms and cancers, such as, for (such as T- modified to assess the ability of example, leukemia, lymphoma (e.g., T cell cells). polypeptides of the invention lymphoma, Burkitt's lymphoma, non- (including antibodies and Hodgkins lymphoma, Hodgkin's disease), agonists or antagonists of the melanoma, and prostate, breast, lung, invention) to regulate STAT colon, pancreatic, esophageal, stomach, transcription factors and brain, liver and urinary cancer. Other modulate gene expression preferred indications include benign involved in a wide variety of dysproliferative disorders and pre- cell functions. Exemplary neoplastic conditions, such as, for example, assays for transcription through hyperplasia, metaplasia, and/or dysplasia. the GAS response element that Preferred indications include autoimmune may be used or routinely diseases (e.g., rheumatoid arthritis, modified to test GAS-response systemic lupus erythematosis, multiple element activity of polypeptides sclerosis and/or as described below), of the invention (including immunodeficiencies (e.g., as described antibodies and agonists or below), boosting a T cell-mediated immune antagonists of the invention) response, and suppressing a T cell- include assays disclosed in mediated immune response. Additional Berger et al., Gene 66: 1-10 preferred indications include inflammation (1998); Cullen and Malm, and inflammatory disorders. Highly Methods in Enzymol 216: 362-368 preferred indications include blood (1992); Henthorn et al., disorders (e.g., as described below under Proc Natl Acad Sci USA “Immune Activity”, “Blood-Related 85: 6342-6346 (1988); Disorders”, and/or ““Cardiovascular Matikainen et al., Blood Disorders””), and infection (e.g., viral 93(6): 1980-1991 (1999); and infections, tuberculosis, infections Henttinen et al., J Immunol associated with chronic granulomatosus 155(10): 4582-4587 (1995), the disease and malignant osteoporosis, and/or contents of each of which are an infectious disease as described below herein incorporated by under “Infectious Disease”). An additional reference in its entirety. preferred indication is idiopathic Exemplary human T cells, such pulmonary fibrosis. Preferred as the MOLT4 cell line, that indications include anemia, pancytopenia, may be used according to these leukopenia, thrombocytopenia, acute assays are publicly available lymphocytic anemia (ALL), (e.g., through the ATCC ™). plasmacytomas, multiple myeloma, arthritis, AIDS, granulomatous disease, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and asthma and allergy. 13 HTADX17 91 Activation of Assays for the activation of Highly preferred indications include transcription transcription through the NFKB inflammation and inflammatory disorders. through NFKB response element are well- Highly preferred indications include blood response known in the art and may be disorders (e.g., as described below under element in used or routinely modified to “Immune Activity”, “Blood-Related immune cells assess the ability of Disorders”, and/or ““Cardiovascular (such as T- polypeptides of the invention Disorders””). Highly preferred indications cells). (including antibodies and include autoimmune diseases (e.g., agonists or antagonists of the rheumatoid arthritis, systemic lupus invention) to regulate NFKB erythematosis, multiple sclerosis and/or as transcription factors and described below), and immunodeficiencies modulate expression of (e.g., as described below). An additional immunomodulatory genes. highly preferred indication is infection Exemplary assays for (e.g., AIDS, and/or an infectious disease as transcription through the NFKB described below under “Infectious response element that may be Disease”). Highly preferred used or rountinely modified to indications include neoplastic diseases test NFKB-response element (e.g., melanoma, leukemia, lymphoma, activity of polypeptides of the and/or as described below under invention (including antibodies “Hyperproliferative Disorders”). Highly and agonists or antagonists of preferred indications include neoplasms the invention) include assays and cancers, such as, for example, disclosed in Berger et al., Gene melanoma, renal cell carcinoma, leukemia, 66: 1-10 (1998); Cullen and lymphoma, and prostate, breast, lung, Malm, Methods in Enzymol colon, pancreatic, esophageal, stomach, 216: 362-368 (1992); Henthorn brain, liver and urinary cancer. Other et al., Proc Natl Acad Sci USA preferred indications include benign 85: 6342-6346 (1988); Black et dysproliferative disorders and pre- al., Virus Gnes 15(2): 105-117 neoplastic conditions, such as, for example, (1997); and Fraser et al., hyperplasia, metaplasia, and/or dysplasia. 29(3): 838-844 (1999), the Preferred indications also include anemia, contents of each of which are pancytopenia, leukopenia, herein incorporated by thrombocytopenia, Hodgkin's disease, reference in its entirety. acute lymphocytic anemia (ALL), Exemplary human T cells, such plasmacytomas, multiple myeloma, as the MOLT4, that may be Burkitt's lymphoma, arthritis, AIDS, used according to these assays granulomatous disease, inflammatory are publicly available (e.g., bowel disease, sepsis, neutropenia, through the ATCC ™). neutrophilia, psoriasis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, suppression of immune reactions to transplanted organs, asthma and allergy. 13 HTADX17 93 Activation of Assays for the activation of Highly preferred indications include transcription transcription through the blood disorders (e.g., as described below through NFAT Nuclear Factor of Activated T under “Immune Activity”, “Blood-Related response in cells (NFAT) response element Disorders”, and/or “Cardiovascular immune cells are well-known in the art and Disorders”). Highly preferred indications (such as T- may be used or routinely include autoimmune diseases (e.g., cells). modified to assess the ability of rheumatoid arthritis, systemic lupus polypeptides of the invention erythematosis, multiple sclerosis and/or as (including antibodies and described below), immunodeficiencies agonists or antagonists of the (e.g., as described below), boosting a T invention) to regulate NFAT cell-mediated immune response, and transcription factors and suppressing a T cell-mediated immune modulate expression of genes response. Additional highly preferred involved in immunomodulatory indications include inflammation and functions. Exemplary assays inflammatory disorders. An additional for transcription through the highly preferred indication is infection NFAT response element that (e.g., an infectious disease as described may be used or routinely below under “Infectious Disease”). modified to test NFAT- Preferred indications include neoplastic response element activity of diseases (e.g., leukemia, lymphoma, and/or polypeptides of the invention as described below under (including antibodies and “Hyperproliferative Disorders”). Preferred agonists or antagonists of the indications include neoplasms and cancers, invention) include assays such as, for example, leukemia, lymphoma, disclosed in Berger et al., Gene and prostate, breast, lung, colon, 66: 1-10 (1998); Cullen and pancreatic, esophageal, stomach, brain, Malm, Methods in Enzymol liver and urinary cancer. Other preferred 216: 362-368 (1992); Henthorn indications include benign dysproliferative et al., Proc Natl Acad Sci USA disorders and pre-neoplastic conditions, 85: 6342-6346 (1988); Serfling such as, for example, hyperplasia, et al., Biochim Biophys Acta metaplasia, and/or dysplasia. Preferred 1498(1): 1-18 (2000); De Boer indications also include anemia, et al., Int J Biochem Cell Biol pancytopenia, leukopenia, 31(10): 1221-1236 (1999); thrombocytopenia, Hodgkin's disease, Fraser et al., Eur J Immunol acute lymphocytic anemia (ALL), 29(3): 838-844 (1999); and plasmacytomas, multiple myeloma, Yeseen et al., J Biol Chem Burkitt's lymphoma, arthritis, AIDS, 268(19): 14285-14293 (1993), granulomatous disease, inflammatory the contents of each of which bowel disease, sepsis, neutropenia, are herein incorporated by neutrophilia, psoriasis, suppression of reference in its entirety. T cells immune reactions to transplanted organs that may be used according to and tissues, hemophilia, hypercoagulation, these assays are publicly diabetes mellitus, endocarditis, meningitis, available (e.g., through the Lyme Disease, asthma and allergy. ATCC ™). Exemplary human T cells that may be used according to these assays include the JURKAT cell line, which is a suspension culture of leukemia cells that produce IL- 2 when stimulated. 13 HTADX17 93 Activation of Assays for the activation of Highly preferred indications include transcription transcription through the neoplastic diseases (e.g., leukemia, through GAS Gamma Interferon Activation lymphoma, and/or as described below response Site (GAS) response element under “Hyperproliferative Disorders”). element in are well-known in the art and Highly preferred indications include immune cells may be used or routinely neoplasms and cancers, such as, for (such as T- modified to assess the ability of example, leukemia, lymphoma (e.g., T cell cells). polypeptides of the invention lymphoma, Burkitt's lymphoma, non- (including antibodies and Hodgkins lymphoma, Hodgkin″s disease), agonists or antagonists of the melanoma, and prostate, breast, lung, invention) to regulate STAT colon, pancreatic, esophageal, stomach, transcription factors and brain, liver and urinary cancer. Other modulate gene expression preferred indications include benign involved in a wide variety of dysproliferative disorders and pre- cell functions. Exemplary neoplastic conditions, such as, for example, assays for transcription through hyperplasia, metaplasia, and/or dysplasia. the GAS response element that Preferred indications include autoimmune may be used or routinely diseases (e.g., rheumatoid arthritis, modified to test GAS-response systemic lupus erythematosis, multiple element activity of polypeptides sclerosis and/or as described below), of the invention (including immunodeficiencies (e.g., as described antibodies and agonists or below), boosting a T cell-mediated immune antagonists of the invention) response, and suppressing a T cell- include assays disclosed in mediated immune response. Additional Berger et al., Gene 66: 1-10 preferred indications include inflammation (1998); Cullen and Malm, and inflammatory disorders. Highly Methods in Enzymol 216: 362-368 preferred indications include blood (1992); Henthorn et al., disorders (e.g., as described below under Proc Natl Acad Sci USA “Immune Activity”, “Blood-Related 85: 6342-6346 (1988); Disorders”, and/or “Cardiovascular Matikainen et al., Blood Disorders”), and infection (e.g., viral 93(6): 1980-1991 (1999); and infections, tuberculosis, infections Henttinen et al., J Immunol associated with chronic granulomatosus 155(10): 4582-4587 (1995), the disease and malignant osteoporosis, and/or contents of each of which are an infectious disease as described below herein incorporated by under “Infectious Disease”). An additional reference in its entirety. preferred indication is idiopathic Exemplary human T cells, such pulmonary fibrosis. Preferred as the MOLT4 cell line, that indications include anemia, pancytopenia, may be used according to these leukopenia, thrombocytopenia, acute assays are publicly available lymphocytic anemia (ALL), (e.g., through the ATCC ™). plasmacytomas, multiple myeloma, arthritis, AIDS, granulomatous disease, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and asthma and allergy. 13 HTADX17 93 Activation of Assays for the activation of Highly preferred indications include transcription transcription through the NFKB inflammation and inflammatory disorders. through NFKB response element are well- Highly preferred indications include blood response known in the art and may be disorders (e.g., as described below under element in used or routinely modified to “Immune Activity”, “Blood-Related immune cells assess the ability of Disorders”, and/or “Cardiovascular (such as T- polypeptides of the invention Disorders”). Highly preferred indications cells). (including antibodies and include autoimmune diseases (e.g., agonists or antagonists of the rheumatoid arthritis, systemic lupus invention) to regulate NFKB erythematosis, multiple sclerosis and/or as transcription factors and described below), and immunodeficiencies modulate expression of (e.g., as described below). An additional immunomodulatory genes. highly preferred indication is infection Exemplary assays for (e.g., AIDS, and/or an infectious disease as transcription through the NFKB described below under “Infectious response element that may be Disease”). Highly preferred used or rountinely modified to indications include neoplastic diseases test NFKB-response element (e.g., melanoma, leukemia, lymphoma, activity of polypeptides of the and/or as described below under invention (including antibodies “Hyperproliferative Disorders”). Highly and agonists or antagonists of preferred indications include neoplasms the invention) include assays and cancers, such as, for example, disclosed in Berger et al., Gene melanoma, renal cell carcinoma, leukemia, 66: 1-10 (1998); Cullen and lymphoma, and prostate, breast, lung, Malm, Methods in Enzymol colon, pancreatic, esophageal, stomach, 216: 362-368 (1992); Henthorn brain, liver and urinary cancer. Other et al., Proc Natl Acad Sci USA preferred indications include benign 85: 6342-6346 (1988); Black et dysproliferative disorders and pre- al., Virus Gnes 15(2): 105-117 neoplastic conditions, such as, for example, (1997); and Fraser et al., hyperplasia, metaplasia, and/or dysplasia. 29(3): 838-844 (1999), the Preferred indications also include anemia, contents of each of which are pancytopenia, leukopenia, herein incorporated by thrombocytopenia, Hodgkin's disease, reference in its entirety. acute lymphocytic anemia (ALL), Exemplary human T cells, such plasmacytomas, multiple myeloma, as the MOLT4, that may be Burkitt's lymphoma, arthritis, AIDS, used according to these assays granulomatous disease, inflammatory are publicly available (e.g., bowel disease, sepsis, neutropenia, through the ATCC ™). neutrophilia, psoriasis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, suppression of immune reactions to transplanted organs, asthma and allergy. 13 HTADX17 93 IL-8 in Normal Human Bronchial Epitheliae 14 HJACG02 105 MIP-1a in HMC 14 HJACG02 105 Proliferation of Assays for the regulation (i.e. pre-adipose increases or decreases) of cells (such as viability and proliferation of 3T3-L1 cells) cells in vitro are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pre-adipose cells and cell lines. For example, the CellTiter- Gloô Luminescent Cell Viability Assay (PROMEGA ™ Corp., Madison, WI, USA) can be used to measure the number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. 3T3-L1 is a mouse preadipocyte cell line. It is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation. Cells were differentiated to an adipose-like state before being used in the screen. See Green H and Meuth M., Cell 3: 127-133 (1974), which is herein incorporated by reference in its entirety. 14 HJACG02 105 MCP-1 in HUVEC 14 HJACG02 105 Activation or This reporter assay measures inhibition of activation or inhibition of the transcription NFkB signaling pathway in through NFKB Ku812 human basophil cell response line. Assays for the activation element in or inhibition of transcription immune cells through the NFKB response (such as element are well-known in the basophils). art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate NFKB transcription factors and modulate expression of immunomodulatory genes. NFkB is important in the pathogenesis of asthma. Exemplary assays for transcription through the NFKB response element that may be used or rountinely modified to test NFKB-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Marone et al, Int Arch Allergy Immunol 114(3): 207-17 (1997), the contents of each of which are herein incorporated by reference in its entirety. Cells were pretreated with SID supernatants or controls for 15-18 hours, and then 10 ng/mL of TNF was added to stimulate the NFkB reporter. SEAP activity was measured after 48 hours. Basophils that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary human basophil cell lines that may be used according to these assays include Ku812, originally established from a patient with chronic myelogenous leukemia. It is an immature prebasophilic cell line that can be induced to differentiate into mature basophils. See, Kishi et al., Leuk Res. 9: 381-390 (1985); Blom et al., Eur J Immunol. 22: 2025-32 (1992), where the contents of each are herein incorporated by reference in its entirety. 16 HSVAK93 121 Production of Assays for measuring Highly preferred indications include VCAM in expression of VCAM are well- inflammation (acute and chronic), endothelial known in the art and may be restnosis, atherosclerosis, asthma and cells (such as used or routinely modified to allergy. Highly preferred indications human assess the ability of include inflammation and inflammatory umbilical vein polypeptides of the invention disorders, immunological disorders, endothelial (including antibodies and neoplastic disorders (e.g. cells agonists or antagonists of the cancer/tumorigenesis), and cardiovascular (HUVEC)) invention) to regulate VCAM disorders (such as described below under expression. For example, “Immune Activity”, “Blood-Related FMAT may be used to meaure Disorders”, “Hyperproliferative Disorders” the upregulation of cell surface and/or “Cardiovascular Disorders”). VCAM-1 expresssion in Highly preferred indications include endothelial cells. Endothelial neoplasms and cancers such as, for cells are cells that line blood example, leukemia, lymphoma, melanoma, vessels, and are involved in renal cell carcinoma, and prostate, breast, functions that include, but are lung, colon, pancreatic, esophageal, not limited to, angiogenesis, stomach, brain, liver and urinary cancer. vascular permeability, vascular Other preferred indications include benign tone, and immune cell dysproliferative disorders and pre- extravasation. Exemplary neoplastic conditions, such as, for example, endothelial cells that may be hyperplasia, metaplasia, and/or dysplasia. used according to these assays include human umbilical vein endothelial cells (HUVEC), which are available from commercial sources. The expression of VCAM (CD106), a membrane-associated protein, can be upregulated by cytokines or other factors, and contributes to the extravasation of lymphocytes, leucocytes and other immune cells from blood vessels; thus VCAM expression plays a role in promoting immune and inflammatory responses. 17 HSDEK49 126 Activation of Assays for the activation of A preferred embodiment of the transcription transcription through the Serum invention includes a method for inhibiting through serum Response Element (SRE) are (e.g., reducing) TNF alpha production. An response well-known in the art and may alternative preferred embodiment of the element in be used or routinely modified to invention includes a method for stimulating immune cells assess the ability of (e.g., increasing) TNF alpha production. (such as T- polypeptides of the invention Preferred indications include blood cells). (including antibodies and disorders (e.g., as described below under agonists or antagonists of the “Immune Activity”, “Blood-Related invention) to regulate the serum Disorders”, and/or “Cardiovascular response factors and modulate Disorders”), Highly preferred indications the expression of genes include autoimmune diseases (e.g., involved in growth. Exemplary rheumatoid arthritis, systemic lupus assays for transcription through erythematosis, Crohn″s disease, multiple the SRE that may be used or sclerosis and/or as described below), routinely modified to test SRE immunodeficiencies (e.g., as described activity of the polypeptides of below), boosting a T cell-mediated immune the invention (including response, and suppressing a T cell- antibodies and agonists or mediated immune response. Additional antagonists of the invention) highly preferred indications include include assays disclosed in inflammation and inflammatory disorders, Berger et al., Gene 66: 1-10 and treating joint damage in patients with (1998); Cullen and Malm, rheumatoid arthritis. An additional highly Methods in Enzymol 216: 362-368 preferred indication is sepsis. Highly (1992); Henthorn et al., preferred indications include neoplastic Proc Natl Acad Sci USA diseases (e.g., leukemia, lymphoma, and/or 85: 6342-6346 (1988); and as described below under Black et al., Virus Genes “Hyperproliferative Disorders”). 12(2): 105-117 (1997), the Additionally, highly preferred indications content of each of which are include neoplasms and cancers, such as, for herein incorporated by example, leukemia, lymphoma, melanoma, reference in its entirety. T cells glioma (e.g., malignant glioma), solid that may be used according to tumors, and prostate, breast, lung, colon, these assays are publicly pancreatic, esophageal, stomach, brain, available (e.g., through the liver and urinary cancer. Other preferred ATCC ™™). Exemplary indications include benign dysproliferative mouse T cells that may be used disorders and pre-neoplastic conditions, according to these assays such as, for example, hyperplasia, include the CTLL cell line, metaplasia, and/or dysplasia. Preferred which is an IL-2 dependent indications include anemia, pancytopenia, suspension culture of T cells leukopenia, thrombocytopenia, Hodgkin's with cytotoxic activity. disease, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, AIDS, granulomatous disease, inflammatory bowel disease, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, cardiac reperfusion injury, and asthma and allergy. An additional preferred indication is infection (e.g., an infectious disease as described below under “Infectious Disease”). 17 HSDEK49 126 Regulation of Assays for the regulation of A highly preferred indication is transcription of transcription of Malic Enzyme diabetes mellitus. An additional Malic Enzyme are well-known in the art and highly preferred indication is a in adipocytes may be used or routinely complication associated with diabetes (e.g., modified to assess the ability of diabetic retinopathy, diabetic nephropathy, polypeptides of the invention kidney disease (e.g., renal failure, (including antibodies and nephropathy and/or other diseases and agonists or antagonists of the disorders as described in the “Renal invention) to regulate Disorders” section below), diabetic transcription of Malic Enzyme, neuropathy, nerve disease and nerve a key enzyme in lipogenesis. damage (e.g., due to diabetic neuropathy), Malic enzyme is involved in blood vessel blockage, heart disease, lipogenesisand its expression is stroke, impotence (e.g., due to diabetic stimulted by insulin. ME neuropathy or blood vessel blockage), promoter contains two direct seizures, mental confusion, drowsiness, repeat (DR1)-like elements nonketotic hyperglycemic-hyperosmolar MEp and MEd identified as coma, cardiovascular disease (e.g., heart putative PPAR response disease, atherosclerosis, microvascular elements. ME promoter may disease, hypertension, stroke, and other also responds to AP1 and other diseases and disorders as described in the transcription factors. “Cardiovascular Disorders” section below), Exemplary assays that may be dyslipidemia, endocrine disorders (as used or routinely modified to described in the “Endocrine Disorders” test for regulation of section below), neuropathy, vision transcription of Malic Enzyme impairment (e.g., diabetic retinopathy and (in adipoocytes) by blindness), ulcers and impaired wound polypeptides of the invention healing, and infection (e.g., infectious (including antibodies and diseases and disorders as described in the agonists or antagonists of the “Infectious Diseases” section below, invention) include assays especially of the urinary tract and skin), disclosed in: Streeper, R. S., et carpal tunnel syndrome and Dupuytren's al., Mol Endocrinol, contracture). An additional highly 12(11): 1778-91 (1998); Garcia- preferred indication is obesity and/or Jimenez, C., et al., Mol complications associated with obesity. Endocrinol, 8(10): 1361-9 Additional highly preferred indications (1994); Barroso, I., et al., J Biol include weight loss or alternatively, weight Chem, 274(25): 17997-8004 gain. Aditional highly preferred (1999); Ijpenberg, A., et al., J indications are complications associated Biol Chem, 272(32): 20108-20117 with insulin resistance. (1997); Berger, et al., Gene 66: 1-10 (1988); and, Cullen, B., et al., Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Hepatocytes that may be used according to these assays are publicly available (e.g., through the ATCC ™™) and/or may be routinely generated. Exemplary hepatocytes that may be used according to these assays includes the H4IIE rat liver hepatoma cell line. 17 HSDEK49 126 MIP-1a in HMC 18 HWBAO62 128 Activation of Assays for the activation of A highly preferred embodiment of the transcription transcription through the CD28 invention includes a method for stimulating through CD28 response element are well- T cell proliferation. An alternative highly response known in the art and may be preferred embodiment of the invention element in used or routinely modified to includes a method for inhibiting T cell immune cells assess the ability of proliferation. A highly preferred (such as T- polypeptides of the invention embodiment of the invention includes a cells). (including antibodies and method for activating T cells. An agonists or antagonists of the alternative highly preferred embodiment of invention) to stimulate IL-2 the invention includes a method for expression in T cells. inhibiting the activation of and/or Exemplary assays for inactivating T cells. A highly preferred transcription through the CD28 embodiment of the invention includes a response element that may be method for stimulating (e.g., increasing) used or routinely modified to IL-2 production. An alternative highly test CD28-response element preferred embodiment of the invention activity of polypeptides of the includes a method for inhibiting (e.g., invention (including antibodies reducing) IL-2 production. Additional and agonists or antagonists of highly preferred indications include the invention) include assays inflammation and inflammatory disorders. disclosed in Berger et al., Gene Highly preferred indications include 66: 1-10 (1998); Cullen and autoimmune diseases (e.g., rheumatoid Malm, Methods in Enzymol arthritis, systemic lupus erythematosis, 216: 362-368 (1992); Henthorn multiple sclerosis and/or as described et al., Proc Natl Acad Sci USA below), immunodeficiencies (e.g., as 85: 6342-6346 (1988); McGuire described below), boosting a T cell- and Iacobelli, J Immunol mediated immune response, and 159(3): 1319-1327 (1997); Parra suppressing a T cell-mediated immune et al., J Immunol 166(4): 2437-2443 response. Highly preferred indications (2001); and Butscher et include neoplastic diseases (e.g., al., J Biol Chem 3(1): 552-560 melanoma, renal cell carcinoma, leukemia, (1998), the contents of each of lymphoma, and/or as described below which are herein incorporated under “Hyperproliferative Disorders”). by reference in its entirety. T Highly preferred indications include cells that may be used neoplasms and cancers, such as, for according to these assays are example, melanoma (e.g., metastatic publicly available (e.g., through melanoma), renal cell carcinoma (e.g., the ATCC ™). Exemplary metastatic renal cell carcinoma), leukemia, human T cells that may be used lymphoma (e.g,. T cell lymphoma), and according to these assays prostate, breast, lung, colon, pancreatic, include the SUPT cell line, esophageal, stomach, brain, liver and which is a suspension culture of urinary cancer. Other preferred indications IL-2 and IL-4 responsive T include benign dysproliferative disorders cells. and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. A highly preferred indication includes infection (e.g., AIDS, tuberculosis, infections associated with granulomatous disease, and osteoporosis, and/or as described below under “Infectious Disease”). A highly preferred indication is AIDS. Additional highly preferred indications include suppression of immune reactions to transplanted organs and/or tissues, uveitis, psoriasis, and tropical spastic paraparesis. Preferred indications include blood disorders (e.g., as described below under “Immune Activity”, “Blood-Related Disorders”, and/or “Cardiovascular Disorders”). Preferred indications also include anemia, pancytopenia, leukopenia, thrombocytopenia, Hodgkin's disease, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, granulomatous disease, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, asthma and allergy. 18 HWBAO62 128 Caspase (+paclitaxel) in SW480 19 HWHGU54 132 Activation of Assays for the activation of Preferred indications include blood transcription transcription through the cAMP disorders (e.g., as described below under through cAMP response element are well- “Immune Activity”, “Blood-Related response known in the art and may be Disorders”, and/or “Cardiovascular element in used or routinely modified to Disorders”), and infection (e.g., an immune cells assess the ability of infectious disease as described below under (such as T- polypeptides of the invention “Infectious Disease”). Preferred cells). (including antibodies and indications include autoimmune diseases agonists or antagonists of the (e.g., rheumatoid arthritis, systemic lupus invention) to increase cAMP erythematosis, multiple sclerosis and/or as and regulate CREB described below), immunodeficiencies transcription factors, and (e.g., as described below), boosting a T modulate expression of genes cell-mediated immune response, and involved in a wide variety of suppressing a T cell-mediated immune cell functions. Exemplary response. Additional preferred indications assays for transcription through include inflammation and inflammatory the cAMP response element disorders. Highly preferred indications that may be used or routinely include neoplastic diseases (e.g., leukemia, modified to test cAMP- lymphoma, and/or as described below response element activity of under “Hyperproliferative Disorders”). polypeptides of the invention Highly preferred indications include (including antibodies and neoplasms and cancers, such as, for agonists or antagonists of the example, leukemia, lymphoma (e.g., T cell invention) include assays lymphoma, Burkitt's lymphoma, non- disclosed in Berger et al., Gene Hodgkins lymphoma, Hodgkin″s disease), 66: 1-10 (1998); Cullen and melanoma, and prostate, breast, lung, Malm, Methods in Enzymol colon, pancreatic, esophageal, stomach, 216: 362-368 (1992); Henthorn brain, liver and urinary cancer. Other et al., Proc Natl Acad Sci USA preferred indications include benign 85: 6342-6346 (1988); Black et dysproliferative disorders and pre- al., Virus Genes 15(2): 105-117 neoplastic conditions, such as, for example, (1997); and Belkowski et al., J hyperplasia, metaplasia, and/or dysplasia. Immunol 161(2): 659-665 Preferred indications include anemia, (1998), the contents of each of pancytopenia, leukopenia, which are herein incorporated thrombocytopenia, acute lymphocytic by reference in its entirety. T anemia (ALL), plasmacytomas, multiple cells that may be used myeloma, arthritis, AIDS, granulomatous according to these assays are disease, inflammatory bowel disease, publicly available (e.g., through sepsis, neutropenia, neutrophilia, psoriasis, the ATCC ™). Exemplary suppression of immune reactions to mouse T cells that may be used transplanted organs and tissues, according to these assays hemophilia, hypercoagulation, diabetes include the CTLL cell line, mellitus, endocarditis, meningitis, Lyme which is a suspension culture of Disease, and asthma and allergy. IL-2 dependent cytotoxic T cells. 19 HWHGU54 132 Production of Assays measuring production Highly preferred indications include IL-8 by by of IL-8 are well known in the immunological and inflammatory disorders endothelial art and may be used or (e.g., such as allergy, asthma, leukemia, cells (such as routinely modified to assess the etc. and as described below under “Immune Human ability of polypeptides of the Activity”, and “Blood-Related Disorders”). Umbilical Cord invention (including antibodies Highly preferred indications also includie Endothelial and agonists or antagonists of autoimmune disorders (e.g., rheumatoid Cells). the invention) to regulate arthritis, systemic lupus erythematosis, production and/or secretion of Crohn″s disease, multiple sclerosis and/or IL-8. For example, FMAT may as described below), neoplastic disorders be used or routinely modified to (e.g., organ cancers such as lung, liver, assess the ability of colon cancer, and/or as described below polypeptides of the invention under “Hyperproliferative Disorders”), and (including antibodies and cardiovascular disorders (e.g. such as agonists or antagonists of the described below under “Cardiovascular invention) to regulate Disorders”). Preferred indications include production and/or secretion of thrombosis, bacteremia and sepsis IL-8 from endothelial cells syndrome and consequent complications (such as human umbilical vein (such as acute respiratory distress endothelial cells (HUVEC)). syndrome and systemic ischemia- HUVECs are endothelial cells reperfusion resulting from septic shock), which line venous blood restnosis and atherosclerosis. vessels, and are involved in functions that include, but are not limited to, angiogenesis, vascular permeability, vascular tone, and immune cell extravasation. Endothelial cells play a pivotal role in the initiation and perpetuation of inflammation and secretion of IL-8 may play an important role in recruitment and activation of immune cells such as neutrophils, macrophages, and lymphocytes. 19 HWHGU54 132 SEAP in HIB/CRE 19 HWHGU54 132 Proliferation of Assays for the regulation (i.e. pre-adipose increases or decreases) of cells (such as viability and proliferation of 3T3-L1 cells) cells in vitro are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pre-adipose cells and cell lines. For example, the CellTiter- Gloô Luminescent Cell Viability Assay (PROMEGA ™ Corp., Madison, WI, USA) can be used to measure the number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. 3T3-L1 is a mouse preadipocyte cell line. It is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation. Cells were differentiated to an adipose-like state before being used in the screen. See Green H and Meuth M., Cell 3: 127-133 (1974), which is herein incorporated by reference in its entirety. 19 HWHGU54 132 MCP-1 in HUVEC 21 HT5GJ57 162 Activation of Assays for the activation of Preferred indications include neoplastic transcription transcription through the AP1 diseases (e.g., as described below under through AP1 response element are known in “Hyperproliferative Disorders”), blood response the art and may be used or disorders (e.g., as described below under element in routinely modified to assess the “Immune Activity”, “Cardiovascular immune cells ability of polypeptides of the Disorders”, and/or “Blood-Related (such as T- invention (including antibodies Disorders”), and infection (e.g., an cells). and agonists or antagonists of infectious disease as described below under the invention) to modulate “Infectious Disease”). Highly preferred growth and other cell functions. indications include autoimmune diseases Exemplary assays for (e.g., rheumatoid arthritis, systemic lupus transcription through the AP1 erythematosis, multiple sclerosis and/or as response element that may be described below) and immunodeficiencies used or routinely modified to (e.g., as described below). Additional test AP1-response element highly preferred indications include activity of polypeptides of the inflammation and inflammatory disorders. invention (including antibodies Highly preferred indications also include and agonists or antagonists of neoplastic diseases (e.g., leukemia, the invention) include assays lymphoma, and/or as described below disclosed in Berger et al., Gene under “Hyperproliferative Disorders”). 66: 1-10 (1988); Cullen and Highly preferred indications include Malm, Methods in Enzymol neoplasms and cancers, such as, leukemia, 216: 362-368 (1992); Henthorn lymphoma, prostate, breast, lung, colon, et al., Proc Natl Acad Sci USA pancreatic, esophageal, stomach, brain, 85: 6342-6346 (1988); Rellahan liver, and urinary cancer. Other preferred et al., J Biol Chem indications include benign dysproliferative 272(49): 30806-30811 (1997); disorders and pre-neoplastic conditions, Chang et al., Mol Cell Biol such as, for example, hyperplasia, 18(9): 4986-4993 (1998); and metaplasia, and/or dysplasia. Preferred Fraser et al., Eur J Immunol indications include arthritis, asthma, AIDS, 29(3): 838-844 (1999), the allergy, anemia, pancytopenia, leukopenia, contents of each of which are thrombocytopenia, Hodgkin's disease, herein incorporated by acute lymphocytic anemia (ALL), reference in its entirety. T cells plasmacytomas, multiple myeloma, that may be used according to Burkitt's lymphoma, granulomatous these assays are publicly disease, inflammatory bowel disease, available (e.g., through the sepsis, psoriasis, suppression of immune ATCC ™). Exemplary mouse T reactions to transplanted organs and tissues, cells that may be used endocarditis, meningitis, and Lyme according to these assays Disease. include the CTLL cell line, which is an IL-2 dependent suspension-culture cell line with cytotoxic activity. 21 HT5GJ57 162 Production of MCP-1 FMAT. Assays for A highly preferred embodiment of the MCP-1 immunomodulatory proteins invention includes a method for stimulating that are produced by a large (e.g., increasing) MCP-1 production. An variety of cells and act to alternative highly preferred embodiment of induce chemotaxis and the invention includes a method for activation of monocytes and T inhibiting (e.g., reducing) MCP-1 cells are well known in the art production. A highly preferred indication and may be used or routinely is infection (e.g., an infectious disease as modified to assess the ability of described below under “Infectious polypeptides of the invention Disease”). Additional highly preferred (including antibodies and indications include inflammation and agonists or antagonists of the inflammatory disorders. Preferred invention) to mediate indications include blood disorders (e.g., as immunomodulation, induce described below under “Immune Activity”, chemotaxis, and modulate “Blood-Related Disorders”, and/or immune cell activation. “Cardiovascular Disorders”). Highly Exemplary assays that test for preferred indications include autoimmune immunomodulatory proteins diseases (e.g., rheumatoid arthritis, evaluate the production of cell systemic lupus erythematosis, multiple surface markers, such as sclerosis and/or as described below) and monocyte chemoattractant immunodeficiencies (e.g., as described protein (MCP), and the below). Preferred indications also activation of monocytes and T include anemia, pancytopenia, leukopenia, cells. Such assays that may be thrombocytopenia, Hodgkin's disease, used or routinely modified to acute lymphocytic anemia (ALL), test immunomodulatory and plasmacytomas, multiple myeloma, diffferentiation activity of Burkitt's lymphoma, arthritis, AIDS, polypeptides of the invention granulomatous disease, inflammatory (including antibodies and bowel disease, sepsis, neutropenia, agonists or antagonists of the neutrophilia, psoriasis, suppression of invention) include assays immune reactions to transplanted organs disclosed in Miraglia et al., J and tissues, hemophilia, hypercoagulation, Biomolecular Screening 4: 193-204 diabetes mellitus, endocarditis, meningitis (1999); Rowland et al., (bacterial and viral), Lyme Disease, “Lymphocytes: a practical asthma, and allergy Preferred indications approach” Chapter 6: 138-160 also include neoplastic diseases (e.g., (2000); Satthaporn and Eremin, leukemia, lymphoma, and/or as described J R Coll Surg Ednb 45(1): 9-19 below under “Hyperproliferative (2001); and Verhasselt et al., J Disorders”). Highly preferred indications Immunol 158: 2919-2925 include neoplasms and cancers, such as, (1997), the contents of each of leukemia, lymphoma, prostate, breast, lung, which are herein incorporated colon, pancreatic, esophageal, stomach, by reference in its entirety. brain, liver, and urinary cancer. Other Human dendritic cells that may preferred indications include benign be used according to these dysproliferative disorders and pre- assays may be isolated using neoplastic conditions, such as, for example, techniques disclosed herein or hyperplasia, metaplasia, and/or dysplasia. otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 21 HT5GJ57 162 Inhibition of Reporter Assay: construct squalene contains regulatory and coding synthetase sequence of squalene gene synthetase, the first specific transcription. enzyme in the cholesterol biosynthetic pathway. See Jiang, et al., J. Biol. Chem. 268: 12818-128241(993), the contents of which are herein incorporated by reference in its entirety. Cells were treated with SID supernatants, and SEAP activity was measured after 72 hours. HepG2 is a human hepatocellular carcinoma cell line (ATCC ™ HB-8065). See Knowles et al., Science. 209: 497-9 (1980), the contents of which are herein incorporated by reference in its entirety. 21 HT5GJ57 162 IgG in Human B cells SAC 21 HT5GJ57 162 IL-10 in Human T-cell 2B9 21 HT5GJ57 162 TNFa in Human T-cell 2B9 21 HT5GJ57 162 Caspase (+paclitaxel) in SW480 27 HDHMA45 203 Production of Assays for production of IL-10 Highly preferred indications include allergy IL-10 and and activation of T-cells are and asthma. Additional highly preferred activation of T- well known in the art and may indications include immune and cells. be used or routinely modified to hematopoietic disorders (e.g., as described assess the ability of below under “Immune Activity”, and polypeptides of the invention “Blood-Related Disorders”), autoimmune (including antibodies and diseases (e.g., rheumatoid arthritis, agonists or antagonists of the systemic lupus erythematosis, Crohn″s invention) to stimulate or disease, multiple sclerosis and/or as inhibit production of IL-10 described below), immunodeficiencies and/or activation of T-cells. (e.g., as described below), boosting a T Exemplary assays that may be cell-mediated immune response, and used or routinely modified to suppressing a T cell-mediated immune assess the ability of response. polypeptides and antibodies of the invention (including agonists or antagonists of the invention) to modulate IL-10 production and/or T-cell proliferation include, for example, assays such as disclosed and/or cited in: Robinson, DS, et al., “Th-2 cytokines in allergic disease” Br Med Bull; 56 (4): 956-968 (2000), and Cohn, et al., “T- helper type 2 cell-directed therapy for asthma” Pharmacology & Therapeutics; 88: 187-196 (2000); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL10 secreted from Th2 cells may be measured as a marker of Th2 cell activation. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated via in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 31 HDPPA04 225 Production of MIP-1alpha FMAT. Assays for A highly preferred embodiment of the MIP1alpha immunomodulatory proteins invention includes a method for stimulating produced by activated dendritic MIP1a production. An alternative highly cells that upregulate preferred embodiment of the invention monocyte/macrophage and T includes a method for inhibiting (e.g., cell chemotaxis are well known reducing) MIP1a production. A highly in the art and may be used or preferred indication is infection (e.g., an routinely modified to assess the infectious disease as described below under ability of polypeptides of the “Infectious Disease”). Preferred invention (including antibodies indications include blood disorders (e.g., as and agonists or antagonists of described below under “Immune Activity”, the invention) to mediate “Blood-Related Disorders”, and/or immunomodulation, modulate “Cardiovascular Disorders”). Highly chemotaxis, and modulate T preferred indications include autoimmune cell differentiation. Exemplary diseases (e.g., rheumatoid arthritis, assays that test for systemic lupus erythematosis, multiple immunomodulatory proteins sclerosis and/or as described below) and evaluate the production of immunodeficiencies (e.g., as described chemokines, such as below). Additional highly preferred macrophage inflammatory indications include inflammation and protein 1 alpha (MIP-1a), and inflammatory disorders. Preferred the activation of indications also include anemia, monocytes/macrophages and T pancytopenia, leukopenia, cells. Such assays that may be thrombocytopenia, Hodgkin's disease, used or routinely modified to acute lymphocytic anemia (ALL), test immunomodulatory and plasmacytomas, multiple myeloma, chemotaxis activity of Burkitt's lymphoma, arthritis, AIDS, polypeptides of the invention granulomatous disease, inflammatory (including antibodies and bowel disease, sepsis, neutropenia, agonists or antagonists of the neutrophilia, psoriasis, suppression of invention) include assays immune reactions to transplanted organs disclosed in Miraglia et al., J and tissues, hemophilia, hypercoagulation, Biomolecular Screening 4: 193-204 diabetes mellitus, endocarditis, meningitis, (1999); Rowland et al., Lyme Disease, asthma, and allergy. “Lymphocytes: a practical Preferred indications also include approach” Chapter 6: 138-160 neoplastic diseases (e.g., leukemia, (2000); Satthaporn and Eremin, lymphoma, and/or as described below J R Coll Surg Ednb 45(1): 9-19 under “Hyperproliferative Disorders”). (2001); Drakes et al., Transp Highly preferred indications include Immunol 8(1): 17-29 (2000); neoplasms and cancers, such as, leukemia, Verhasselt et al., J Immunol lymphoma, prostate, breast, lung, colon, 158: 2919-2925 (1997); and pancreatic, esophageal, stomach, brain, Nardelli et al., J Leukoc Biol liver, and urinary cancer. Other preferred 65: 822-828 (1999), the contents indications include benign dysproliferative of each of which are herein disorders and pre-neoplastic conditions, incorporated by reference in its such as, for example, hyperplasia, entirety. Human dendritic cells metaplasia, and/or dysplasia. that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 33 HKABZ65 233 Production of IL-6 FMAT. IL-6 is produced A highly preferred embodiment of the IL-6 by T cells and has strong effects invention includes a method for stimulating on B cells. IL-6 participates in (e.g., increasing) IL-6 production. An IL-4 induced IgE production alternative highly preferred embodiment of and increases IgA production the invention includes a method for (IgA plays a role in mucosal inhibiting (e.g., reducing) IL-6 production. immunity). IL-6 induces A highly preferrred indication is the cytotoxic T cells. Deregulated stimulation or enhancement of mucosal expression of IL-6 has been immunity. Highly preferred indications linked to autoimmune disease, include blood disorders (e.g., as described plasmacytomas, myelomas, and below under “Immune Activity”, “Blood- chronic hyperproliferative Related Disorders”, and/or “Cardiovascular diseases. Assays for Disorders”), and infection (e.g., as immunomodulatory and described below under “Infectious differentiation factor proteins Disease”). Highly preferred indications produced by a large variety of include autoimmune diseases (e.g., cells where the expression level rheumatoid arthritis, systemic lupus is strongly regulated by erythematosis, multiple sclerosis and/or as cytokines, growth factors, and described below) and immunodeficiencies hormones are well known in the (e.g., as described below). Highly art and may be used or preferred indications also include boosting routinely modified to assess the a B cell-mediated immune response and ability of polypeptides of the alternatively suppressing a B cell-mediated invention (including antibodies immune response. Highly preferred and agonists or antagonists of indications include inflammation and the invention) to mediate inflammatory disorders. Additional highly immunomodulation and preferred indications include asthma and differentiation and modulate T allergy. Highly preferred indications cell proliferation and function. include neoplastic diseases (e.g., myeloma, Exemplary assays that test for plasmacytoma, leukemia, lymphoma, immunomodulatory proteins melanoma, and/or as described below evaluate the production of under “Hyperproliferative Disorders”). cytokines, such as IL-6, and the Highly preferred indications include stimulation and upregulation of neoplasms and cancers, such as, myeloma, T cell proliferation and plasmacytoma, leukemia, lymphoma, functional activities. Such melanoma, and prostate, breast, lung, assays that may be used or colon, pancreatic, esophageal, stomach, routinely modified to test brain, liver and urinary cancer. Other immunomodulatory and preferred indications include benign diffferentiation activity of dysproliferative disorders and pre- polypeptides of the invention neoplastic conditions, such as, for example, (including antibodies and hyperplasia, metaplasia, and/or dysplasia. agonists or antagonists of the Preferred indications include anemia, invention) include assays pancytopenia, leukopenia, disclosed in Miraglia et al., J thrombocytopenia, Hodgkin's disease, Biomolecular Screening 4: 193-204 acute lymphocytic anemia (ALL), multiple (1999); Rowland et al., myeloma, Burkitt's lymphoma, arthritis, “Lymphocytes: a practical AIDS, granulomatous disease, approach” Chapter 6: 138-160 inflammatory bowel disease, sepsis, (2000); and Verhasselt et al., J neutropenia, neutrophilia, psoriasis, Immunol 158: 2919-2925 suppression of immune reactions to (1997), the contents of each of transplanted organs and tissues, which are herein incorporated hemophilia, hypercoagulation, diabetes by reference in its entirety. mellitus, endocarditis, meningitis, and Human dendritic cells that may Lyme Disease. An additonal preferred be used according to these indication is infection (e.g., an infectious assays may be isolated using disease as described below under techniques disclosed herein or “Infectious Disease”). otherwise known in the art. Human dendritic cells are antigen presenting cells in suspension culture, which, when activated by antigen and/or cytokines, initiate and upregulate T cell proliferation and functional activities. 33 HKABZ65 233 Activation of Kinase assay. JNK and p38 A highly preferred embodiment of the Endothelial kinase assays for signal invention includes a method for stimulating Cell p38 or transduction that regulate cell endothelial cell growth. An alternative JNK Signaling proliferation, activation, or highly preferred embodiment of the Pathway. apoptosis are well known in the invention includes a method for inhibiting art and may be used or endothelial cell growth. A highly routinely modified to assess the preferred embodiment of the invention ability of polypeptides of the includes a method for stimulating invention (including antibodies endothelial cell proliferation. An alternative and agonists or antagonists of highly preferred embodiment of the the invention) to promote or invention includes a method for inhibiting inhibit cell proliferation, endothelial cell proliferation. A activation, and apoptosis. highly preferred embodiment of the Exemplary assays for JNK and invention includes a method for stimulating p38 kinase activity that may be apoptosis of endothelial cells. An used or routinely modified to alternative highly preferred embodiment of test JNK and p38 kinase- the invention includes a method for induced activity of polypeptides inhibiting (e.g., decreasing) apoptosis of of the invention (including endothelial cells. A highly preferred antibodies and agonists or embodiment of the invention includes a antagonists of the invention) method for stimulating (e.g., increasing) include the assays disclosed in endothelial cell activation. An alternative Forrer et al., Biol Chem 379(8-9): highly preferred embodiment of the 1101-1110 (1998); Gupta et invention includes a method for inhibiting al., Exp Cell Res 247(2): 495-504 (e.g., decreasing) the activation of and/or (1999); Kyriakis JM, inactivating endothelial cells. A Biochem Soc Symp 64: 29-48 highly preferred embodiment of the (1999); Chang and Karin, invention includes a method for stimulating Nature 410(6824): 37-40 angiogenisis. An alternative highly (2001); and Cobb MH, Prog preferred embodiment of the invention Biophys Mol Biol 71(3-4): 479-500 includes a method for inhibiting (1999); the contents of angiogenesis. A highly preferred each of which are herein embodiment of the invention includes a incorporated by reference in its method for reducing cardiac hypertrophy. entirety. Endothelial cells that An alternative highly preferred may be used according to these embodiment of the invention includes a assays are publicly available method for inducing cardiac hypertrophy. (e.g., through the ATCC ™). Highly preferred indications include Exemplary endothelial cells neoplastic diseases (e.g., as described that may be used according to below under “Hyperproliferative these assays include human Disorders”), and disorders of the umbilical vein endothelial cells cardiovascular system (e.g., heart disease, (HUVEC), which are congestive heart failure, hypertension, endothelial cells which line aortic stenosis, cardiomyopathy, valvular venous blood vessels, and are regurgitation, left ventricular dysfunction, involved in functions that atherosclerosis and atherosclerotic vascular include, but are not limited to, disease, diabetic nephropathy, intracardiac angiogenesis, vascular shunt, cardiac hypertrophy, myocardial permeability, vascular tone, and infarction, chronic hemodynamic overload, immune cell extravasation. and/or as described below under “Cardiovascular Disorders”). Highly preferred indications include cardiovascular, endothelial and/or angiogenic disorders (e.g., systemic disorders that affect vessels such as diabetes mellitus, as well as diseases of the vessels themselves, such as of the arteries, capillaries, veins and/or lymphatics). Highly preferred are indications that stimulate angiogenesis and/or cardiovascularization. Highly preferred are indications that inhibit angiogenesis and/or cardiovascularization. Highly preferred indications include antiangiogenic activity to treat solid tumors, leukemias, and Kaposi″s sarcoma, and retinal disorders. Highly preferred indications include neoplasms and cancer, such as, Kaposi″s sarcoma, hemangioma (capillary and cavernous), glomus tumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma, angiosarcoma, haemangiopericytoma, lymphangioma, lymphangiosarcoma. Highly preferred indications also include cancers such as, prostate, breast, lung, colon, pancreatic, esophageal, stomach, brain, liver, and urinary cancer. Preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Highly preferred indications also include arterial disease, such as, atherosclerosis, hypertension, coronary artery disease, inflammatory vasculitides, Reynaud″s disease and Reynaud″s phenomenom, aneurysms, restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular disorders such as peripheral vascular disease, and cancer. Highly preferred indications also include trauma such as wounds, burns, and injured tissue (e.g., vascular injury such as, injury resulting from balloon angioplasty, and atheroschlerotic lesions), implant fixation, scarring, ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal diseases such as acute renal failure, and osteoporosis. Additional highly preferred indications include stroke, graft rejection, diabetic or other retinopathies, thrombotic and coagulative disorders, vascularitis, lymph angiogenesis, sexual disorders, age-related macular degeneration, and treatment/ prevention of endometriosis and related conditions. Additional highly preferred indications include fibromas, heart disease, cardiac arrest, heart valve disease, and vascular disease. Preferred indications include blood disorders (e.g., as described below under “Immune Activity”, “Blood- Related Disorders”, and/or “Cardiovascular Disorders”). Preferred indications include autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis and/or as described below) and immunodeficiencies (e.g., as described below). Additional preferred indications include inflammation and inflammatory disorders (such as acute and chronic inflammatory diseases, e.g., inflammatory bowel disease and Crohn's disease), and pain management. 33 HKABZ65 233 Regulation of Caspase Apoptosis. Assays A highly preferred indication is apoptosis in for caspase apoptosis are well diabetes mellitus. An additional pancreatic beta known in the art and may be highly preferred indication is a cells. used or routinely modified to complication associated with diabetes (e.g., assess the ability of diabetic retinopathy, diabetic nephropathy, polypeptides of the invention kidney disease (e.g., renal failure, (including antibodies and nephropathy and/or other diseases and agonists or antagonists of the disorders as described in the “Renal invention) to promote caspase Disorders” section below), diabetic protease-mediated apoptosis. neuropathy, nerve disease and nerve Apoptosis in pancreatic beta is damage (e.g., due to diabetic neuropathy), associated with induction and blood vessel blockage, heart disease, progression of diabetes. stroke, impotence (e.g., due to diabetic Exemplary assays for caspase neuropathy or blood vessel blockage), apoptosis that may be used or seizures, mental confusion, drowsiness, routinely modified to test nonketotic hyperglycemic-hyperosmolar capase apoptosis activity of coma, cardiovascular disease (e.g., heart polypeptides of the invention disease, atherosclerosis, microvascular (including antibodies and disease, hypertension, stroke, and other agonists or antagonists of the diseases and disorders as described in the invention) include the assays “Cardiovascular Disorders” section below), disclosed in: Loweth, AC, et dyslipidemia, endocrine disorders (as al., FEBS Lett, 400(3): 285-8 described in the “Endocrine Disorders” (1997); Saini, KS, et al., section below), neuropathy, vision Biochem Mol Biol Int, impairment (e.g., diabetic retinopathy and 39(6): 1229-36 (1996); blindness), ulcers and impaired wound Krautheim, A., et al., Br J healing, and infection (e.g., infectious Pharmacol, 129(4): 687-94 diseases and disorders as described in the (2000); Chandra J, et al., “Infectious Diseases” section below, Diabetes, 50 Suppl 1: S44-7 especially of the urinary tract and skin), (2001); Suk K, et al., J carpal tunnel syndrome and Dupuytren's Immunol, 166(7): 4481-9 contracture). An additional highly (2001); Tejedo J, et al., FEBS preferred indication is obesity and/or Lett, 459(2): 238-43 (1999); complications associated with obesity. Zhang, S., et al., FEBS Lett, Additional highly preferred indications 455(3): 315-20 (1999); Lee et include weight loss or alternatively, weight al., FEBS Lett 485(2-3): 122-126 gain. Aditional highly preferred (2000); Nor et al., J Vasc indications are complications associated Res 37(3): 209-218 (2000); and with insulin resistance. Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Pancreatic cells that may be used according to these assays are publicly available (e.g., through the ATCC ™) and/or may be routinely generated. Exemplary pancreatic cells that may be used according to these assays include RIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell line derived from a radiation induced transplantable rat islet cell tumor. The cells produce and secrete islet polypeptide hormones, and produce insulin, somatostatin, and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519.

Tables 1E.1 and 1E.2 provide information related to biological activities for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Tables 1E.2 also provide information related to assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column of Table 1E.1 (“Gene No.”) provides the gene number in the application for each clone identifier. The second column of Table 1E.1 (“cDNA Clone ID:”) provides the unique clone identifier for each clone as previously described and indicated in Table 1A through Table 1D. The third column of Table 1E.1 (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A and 1B.1). The fourth column of Table 1E.1 (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides).

In Table 1E.2, each of the biological activities of Table 1E.1 are listed by “Biological Activity Number” and the corresponding “Biological Activity” and are followed by an “Exemplary Activity Assay” column and a “Preferred Indication” column; however, for some biological activities no “Exemplary Activity Assay” or “Preferred Indication” is given. The “Exemplary Activity Assay” column describes the biological activity listed in the column that precedes it and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The “Preferred Indication” column also refers to the biological activity listed in the preceding column and describes disease(s) or disorder(s) that may be detected, diagnosed, prevented, treated, or ameliorated by the nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof).

Tables 1E, 1E.1, and 1E.2 describe the use of, inter alia, FMAT technology for testing or demonstrating various biological activities. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based system which provides a means to perform nonradioactive cell- and bead-based assays to detect activation of cell signal transduction pathways. This technology was designed specifically for ligand binding and immunological assays. Using this technology, fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using a data processing system. Unbound fluorophore comprising the background signal is ignored, allowing for a wide variety of homogeneous assays. FMAT technology may be used for peptide ligand binding assays, immunofluorescence, apoptosis, cytotoxicity, and bead-based immunocapture assays. See, Miraglia S et. al., “Homogeneous cell and bead based assays for high throughput screening using fluorometric microvolume assay technology,” Journal of Biomolecular Screening; 4:193-204 (1999). In particular, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides (including polypeptide fragments and variants) to activate signal transduction pathways. For example, FMAT technology may be used to test, confirm, and/or identify the ability of polypeptides to upregulate production of immunomodulatory proteins (such as, for example, interleukins, GM-CSF, Rantes, and Tumor Necrosis factors, as well as other cellular regulators (e.g. insulin)).

Tables 1E, 1E.1, and 1E.2 also describe the use of kinase assays for testing, demonstrating, or quantifying biological activity. In this regard, the phosphorylation and de-phosphorylation of specific amino acid residues (e.g. Tyrosine, Serine, Threonine) on cell-signal transduction proteins provides a fast, reversible means for activation and de-activation of cellular signal transduction pathways. Moreover, cell signal transduction via phosphorylation/de-phosphorylation is crucial to the regulation of a wide variety of cellular processes (e.g. proliferation, differentiation, migration, apoptosis, etc.). Accordingly, kinase assays provide a powerful tool useful for testing, confirming, and/or identifying polypeptides (including polypeptide fragments and variants) that mediate cell signal transduction events via protein phosphorylation. See e.g., Forrer, P., Tamaskovic R., and Jaussi, R. “Enzyme-Linked Immunosorbent Assay for Measurement of JNK, ERK, and p38 Kinase Activities” Biol. Chem. 379(8-9): 1101-1110 (1998). Table 1D from U.S. patent application Ser. No. 10/472,532, filed Sep. 20, 2003, is herein incorporated by reference. Table 1D in priority Application No. PCT/US02/09785, filed Mar. 19, 2002, which corresponds to Publication No. WO02/95010, published Nov. 28, 2002 (e.g., pages 238 to 582 of Publication No. WO02/95010) is incorporated by reference herein in its entirety.

TABLE 1E.1 AA SEQ Gene cDNA ID No. Clone ID NO: Y Biological Activity 3 HTEEB42 10 Biological Activity #119: Regulation of transcription of Malic Enzyme in hepatocytes 4 HEQCC55 16 Biological Activity #94: Production of IL-13 and activation of T- cells. 4 HEQCC55 16 Biological Activity #103: Production of MCP-1 11 HLHFP03 79 Biological Activity #12: Activation of T-Cell p38 or JNK Signaling Pathway. 11 HLHFP03 79 Biological Activity #12: Activation of T-Cell p38 or JNK Signaling Pathway. 11 HLHFP03 79 Biological Activity #110: Production of VCAM in endothelial cells (such as human umbilical vein endothelial cells (HUVEC)) 12 HHTLF25 84 Biological Activity #92: Production of IL-10 and downregulation of immune responses 13 HTADX17 93 Biological Activity #21: Activation of transcription through GAS response element in immune cells (such as T-cells). 13 HTADX17 93 Biological Activity #27: Activation of transcription through NFAT response in immune cells (such as T-cells). 13 HTADX17 93 Biological Activity #35: Activation of transcription through NFKB response element in immune cells (such as T-cells). 14 HJACG02 105 Biological Activity #112: Proliferation of pre-adipose cells (such as 3T3-L1 cells) 14 HJACG02 105 Biological Activity #48: Activation or inhibition of transcription through NFKB response element in immune cells (such as basophils). 17 HSDEK49 126 Biological Activity #41: Activation of transcription through serum response element in immune cells (such as T-cells). 17 HSDEK49 126 Biological Activity #118: Regulation of transcription of Malic Enzyme in adipocytes 17 HSDEK49 126 Biological Activity #41: Activation of transcription through serum response element in immune cells (such as T-cells). 17 HSDEK49 126 Biological Activity #118: Regulation of transcription of Malic Enzyme in adipocytes 17 HSDEK49 126 Biological Activity #84: MIP-1a in HMC 18 HWBAO62 128 Biological Activity #17: Activation of transcription through CD28 response element in immune cells (such as T-cells). 19 HWHGU54 132 Biological Activity #16: Activation of transcription through cAMP response element in immune cells (such as T-cells). 19 HWHGU54 132 Biological Activity #100: Production of IL-8 by endothelial cells (such as Human Umbilical Cord Endothelial Cells). 21 HT5GJ57 160 Biological Activity #103: Production of MCP-1 21 HT5GJ57 160 Biological Activity #14: Activation of transcription through AP1 response element in immune cells (such as T-cells). 21 HT5GJ57 162 Biological Activity #14: Activation of transcription through AP1 response element in immune cells (such as T-cells). 21 HT5GJ57 162 Biological Activity #103: Production of MCP-1 27 HDHMA45 203 Biological Activity #91: Production of IL-10 and activation of T- cells. 31 HDPPA04 225 Biological Activity #104: Production of MIP1alpha 33 HKABZ65 233 Biological Activity #5: Activation of Endothelial Cell p38 or JNK Signaling Pathway. 33 HKABZ65 233 Biological Activity #98: Production of IL-6 33 HKABZ65 233 Biological Activity #115: Regulation of apoptosis in pancreatic beta cells. 33 HKABZ65 233 Biological Activity #98: Production of IL-6 33 HKABZ65 233 Biological Activity #5: Activation of Endothelial Cell p38 or JNK Signaling Pathway. 33 HKABZ65 233 Biological Activity #115: Regulation of apoptosis in pancreatic beta cells.

TABLE 1E.2 Biological Activity No. Biological Activity Exemplary Activity Assay Preferred Indication 1 Activation of Kinase assay. Kinase assays, for example A highly preferred embodiment of the invention Adipocyte ERK an Elk-1 kinase assay, for ERK signal includes a method for stimulating adipocyte Signaling Pathway transduction that regulate cell proliferation proliferation. An alternative highly preferred or differentiation are well known in the art embodiment of the invention includes a method and may be used or routinely modified to for inhibiting adipocyte proliferation. A assess the ability of polypeptides of the highly preferred embodiment of the invention invention (including antibodies and agonists includes a method for stimulating adipocyte or antagonists of the invention) to promote differentiation. An alternative highly preferred or inhibit cell proliferation, activation, and embodiment of the invention includes a method differentiation. Exemplary assays for ERK for inhibiting adipocyte differentiation. A kinase activity that may be used or routinely highly preferred embodiment of the invention modified to test ERK kinase-induced includes a method for stimulating (e.g., activity of polypeptides of the invention increasing) adipocyte activation. An alternative (including antibodies and agonists or highly preferred embodiment of the invention antagonists of the invention) include the includes a method for inhibiting the activation assays disclosed in Forrer et al., Biol Chem of (e.g., decreasing) and/or inactivating 379(8-9): 1101-1110 (1998); Le Marchand- adipocytes. Highly preferred indications Brustel Y, Exp Clin Endocrinol Diabetes include endocrine disorders (e.g., as described 107(2): 126-132 (1999); Kyriakis JM, below under “Endocrine Disorders”). Biochem Soc Symp 64: 29-48 (1999); Highly preferred indications also include Chang and Karin, Nature 410(6824): 37-40 neoplastic diseases (e.g., lipomas, liposarcomas, (2001); and Cobb MH, Prog Biophys Mol and/or as described below under Biol 71(3-4): 479-500 (1999); the contents “Hyperproliferative Disorders”). Preferred of each of which are herein incorporated by indications include blood disorders (e.g., reference in its entirety. Mouse adipocyte hypertension, congestive heart failure, blood cells that may be used according to these vessel blockage, heart disease, stroke, assays are publicly available (e.g., through impotence and/or as described below under the ATCC ™). Exemplary mouse adipocyte “Immune Activity”, “Cardiovascular cells that may be used according to these Disorders”, and/or “Blood-Related Disorders”), assays include 3T3-L1 cells. 3T3-L1 is an immune disorders (e.g., as described below adherent mouse preadipocyte cell line that is under “Immune Activity”), neural disorders a continuous substrain of 3T3 fibroblast (e.g., as described below under “Neural Activity cells developed through clonal isolation and and Neurological Diseases”), and infection undergo a pre-adipocyte to adipose-like (e.g., as described below under “Infectious conversion under appropriate differentiation Disease”). A highly preferred conditions known in the art. indication is diabetes mellitus. An additional highly preferred indication is a complication associated with diabetes (e.g., diabetic retinopathy, diabetic nephropathy, kidney disease (e.g., renal failure, nephropathy and/or other diseases and disorders as described in the “Renal Disorders” section below), diabetic neuropathy, nerve disease and nerve damage (e.g., due to diabetic neuropathy), blood vessel blockage, heart disease, stroke, impotence (e.g., due to diabetic neuropathy or blood vessel blockage), seizures, mental confusion, drowsiness, nonketotic hyperglycemic-hyperosmolar coma, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the “Cardiovascular Disorders” section below), dyslipidemia, endocrine disorders (as described in the “Endocrine Disorders” section below), neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, infection (e.g., infectious diseases and disorders as described in the “Infectious Diseases” section below (particularly of the urinary tract and skin). An additional highly preferred indication is obesity and/or complications associated with obesity. Additional highly preferred indications include weight loss or alternatively, weight gain. Additional highly preferred indications are complications associated with insulin resistance. Additional highly preferred indications are disorders of the musculoskeletal systems including myopathies, muscular dystrophy, and/or as described herein. Additional highly preferred indications include, hypertension, coronary artery disease, dyslipidemia, gallstones, osteoarthritis, degenerative arthritis, eating disorders, fibrosis, cachexia, and kidney diseases or disorders. Preferred indications include neoplasms and cancer, such as, lymphoma, leukemia and breast, colon, and kidney cancer. Additional preferred indications include melanoma, prostate, lung, pancreatic, esophageal, stomach, brain, liver, and urinary cancer. Highly preferred indications include lipomas and liposarcomas. Other preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. 2 Activation of Kinase assay. Kinase assays, for example Adipocyte PI3 Kinase an GSK-3 assays, for PI3 kinase signal Signaling Pathway transduction that regulate glucose metabolism and cell survival are well- known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Mouse adipocyte cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 3 Activation of Kinase assay. Kinase assays, for example an A highly preferred embodiment of the invention Endothelial Cell ERK Elk-1 kinase assay, for ERK signal includes a method for stimulating endothelial Signaling Pathway. transduction that regulate cell proliferation cell growth. An alternative highly preferred or differentiation are well known in the art embodiment of the invention includes a method and may be used or routinely modified to for inhibiting endothelial cell growth. A assess the ability of polypeptides of the highly preferred embodiment of the invention invention (including antibodies and agonists includes a method for stimulating endothelial or antagonists of the invention) to promote cell proliferation. An alternative highly or inhibit cell proliferation, activation, and preferred embodiment of the invention includes differentiation. Exemplary assays for ERK a method for inhibiting endothelial cell kinase activity that may be used or routinely proliferation. A highly preferred modified to test ERK kinase-induced embodiment of the invention includes a method activity of polypeptides of the invention for stimulating apoptosis of endothelial cells. (including antibodies and agonists or An alternative highly preferred embodiment of antagonists of the invention) include the the invention includes a method for inhibiting assays disclosed in Forrer et al., Biol Chem (e.g., decreasing) apoptosis of endothelial cells. 379(8-9): 1101-1110 (1998); Berra et al., A highly preferred embodiment of the invention Biochem Pharmacol 60(8): 1171-1178 includes a method for stimulating (e.g., (2000); Gupta et al., Exp Cell Res increasing) endothelial cell activation. An 247(2): 495-504 (1999); Chang and Karin, alternative highly preferred embodiment of the Nature 410(6824): 37-40 (2001); and Cobb invention includes a method for inhibiting the MH, Prog Biophys Mol Biol 71(3-4): 479-500 activation of (e.g., decreasing) and/or (1999); the contents of each of which inactivating endothelial cells. A highly are herein incorporated by reference in its preferred embodiment of the invention includes entirety. Endothelial cells that may be used a method for stimulating endothelial cell according to these assays are publicly differentiation. An alternative highly preferred available (e.g., through the ATCC ™). embodiment of the invention includes a method Exemplary endothelial cells that may be for inhibiting endothelial cell differentiation. used according to these assays include A highly preferred embodiment of the invention human umbilical vein endothelial cells includes a method for stimulating angiogenisis. (HUVEC), which are endothelial cells An alternative highly preferred embodiment of which line venous blood vessels, and are the invention includes a method for inhibiting involved in functions that include, but are angiogenesis. A highly preferred not limited to, angiogenesis, vascular embodiment of the invention includes a method permeability, vascular tone, and immune for reducing cardiac hypertrophy. An cell extravasation. alternative highly preferred embodiment of the invention includes a method for inducing cardiac hypertrophy. Highly preferred indications include neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., heart disease, congestive heart failure, hypertension, aortic stenosis, cardiomyopathy, valvular regurgitation, left ventricular dysfunction, atherosclerosis and atherosclerotic vascular disease, diabetic nephropathy, intracardiac shunt, cardiac hypertrophy, myocardial infarction, chronic hemodynamic overload, and/or as described below under “Cardiovascular Disorders”). Highly preferred indications include cardiovascular, endothelial and/or angiogenic disorders (e.g., systemic disorders that affect vessels such as diabetes mellitus, as well as diseases of the vessels themselves, such as of the arteries, capillaries, veins and/or lymphatics). Highly preferred are indications that stimulate angiogenesis and/or cardiovascularization. Highly preferred are indications that inhibit angiogenesis and/or cardiovascularization. Highly preferred indications include antiangiogenic activity to treat solid tumors, leukemias, and Kaposi's sarcoma, and retinal disorders. Highly preferred indications include neoplasms and cancer, such as, Kaposi's sarcoma, hemangioma (capillary and cavernous), glomus tumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma, angiosarcoma, haemangiopericytoma, lymphangioma, lymphangiosarcoma. Highly preferred indications also include cancers such as, prostate, breast, lung, colon, pancreatic, esophageal, stomach, brain, liver, and urinary cancer. Preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Highly preferred indications also include arterial disease, such as, atherosclerosis, hypertension, coronary artery disease, inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenom, aneurysms, restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular disorders such as peripheral vascular disease, and cancer. Highly preferred indications also include trauma such as wounds, burns, and injured tissue (e.g., vascular injury such as, injury resulting from balloon angioplasty, and atheroschlerotic lesions), implant fixation, scarring, ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal diseases such as acute renal failure, and osteoporosis. Additional highly preferred indications include stroke, graft rejection, diabetic or other retinopathies, thrombotic and coagulative disorders, vascularitis, lymph angiogenesis, sexual disorders, age-related macular degeneration, and treatment/ prevention of endometriosis and related conditions. Additional highly preferred indications include fibromas, heart disease, cardiac arrest, heart valve disease, and vascular disease. Preferred indications include blood disorders (e.g., as described below under “Immune Activity”, “Blood-Related Disorders”, and/or ““Cardiovascular Disorders””). Preferred indications include autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis and/or as described below) and immunodeficiencies (e.g., as described below). Additional preferred indications include inflammation and inflammatory disorders (such as acute and chronic inflammatory diseases, e.g., inflammatory bowel disease and Crohn's disease), and pain management. 4 Activation of Kinase assay. JNK kinase assays for signal A highly preferred embodiment of the invention Endothelial Cell JNK transduction that regulate cell proliferation, includes a method for stimulating endothelial Signaling Pathway. activation, or apoptosis are well known in cell growth. An alternative highly preferred the art and may be used or routinely embodiment of the invention includes a method modified to assess the ability of for inhibiting endothelial cell growth. A polypeptides of the invention (including highly preferred embodiment of the invention antibodies and agonists or antagonists of the includes a method for stimulating endothelial invention) to promote or inhibit cell cell proliferation. An alternative highly proliferation, activation, and apoptosis. preferred embodiment of the invention includes Exemplary assays for JNK kinase activity a method for inhibiting endothelial cell that may be used or routinely modified to proliferation. A highly preferred test JNK kinase-induced activity of embodiment of the invention includes a method polypeptides of the invention (including for stimulating apoptosis of endothelial cells. antibodies and agonists or antagonists of the An alternative highly preferred embodiment of invention) include the assays disclosed in the invention includes a method for inhibiting Forrer et al., Biol Chem 379(8-9): 1101-1110 apoptosis of endothelial cells. A highly (1998); Gupta et al., Exp Cell Res preferred embodiment of the invention includes 247(2): 495-504 (1999); Kyriakis JM, a method for stimulating endothelial cell Biochem Soc Symp 64: 29-48 (1999); activation. An alternative highly preferred Chang and Karin, Nature 410(6824): 37-40 embodiment of the invention includes a method (2001); and Cobb MH, Prog Biophys Mol for inhibiting the activation of and/or Biol 71(3-4): 479-500 (1999); the contents inactivating endothelial cells. A highly of each of which are herein incorporated by preferred embodiment of the invention includes reference in its entirety. Endothelial cells a method for stimulating angiogenisis. An that may be used according to these assays alternative highly preferred embodiment of the are publicly available (e.g., through the invention includes a method for inhibiting ATCC ™). Exemplary endothelial cells that angiogenesis. A highly preferred may be used according to these assays embodiment of the invention includes a method include human umbilical vein endothelial for reducing cardiac hypertrophy. An cells (HUVEC), which are endothelial cells alternative highly preferred embodiment of the which line venous blood vessels, and are invention include a method for inducing cardiac involved in functions that include, but are hypertrophy. Highly preferred indications not limited to, angiogenesis, vascular include neoplastic diseases (e.g., as described permeability, vascular tone, and immune below under “Hyperproliferative Disorders”), cell extravasation. and disorders of the cardiovascular system (e.g., heart disease, congestive heart failure, hypertension, aortic stenosis, cardiomyopathy, valvular regurgitation, left ventricular dysfunction, atherosclerosis and atherosclerotic vascular disease, diabetic nephropathy, intracardiac shunt, cardiac hypertrophy, myocardial infarction, chronic hemodynamic overload, and/or as described below under “Cardiovascular Disorders”). Highly preferred indications include cardiovascular, endothelial and/or angiogenic disorders (e.g., systemic disorders that affect vessels such as diabetes mellitus, as well as diseases of the vessels themselves, such as of the arteries, capillaries, veins and/or lymphatics). Highly preferred are indications that stimulate angiogenesis and/or cardiovascularization. Highly preferred are indications that inhibit angiogenesis and/or cardiovascularization. Highly preferred indications include antiangiogenic activity to treat solid tumors, leukemias, and Kaposi″s sarcoma, and retinal disorders. Highly preferred indications include neoplasms and cancer, such as, Kaposi″s sarcoma, hemangioma (capillary and cavernous), glomus tumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma, angiosarcoma, haemangiopericytoma, lymphangioma, lymphangiosarcoma. Highly preferred indications also include cancers such as, prostate, breast, lung, colon, pancreatic, esophageal, stomach, brain, liver, and urinary cancer. Preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Highly preferred indications also include arterial disease, such as, atherosclerosis, hypertension, coronary artery disease, inflammatory vasculitides, Reynaud″s disease and Reynaud″s phenomenom, aneurysms, restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular disorders such as peripheral vascular disease, and cancer. Highly preferred indications also include trauma such as wounds, burns, and injured tissue (e.g., vascular injury such as, injury resulting from balloon angioplasty, and atheroschlerotic lesions), implant fixation, scarring, ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal diseases such as acute renal failure, and osteoporosis. Additional highly preferred indications include stroke, graft rejection, diabetic or other retinopathies, thrombotic and coagulative disorders, vascularitis, lymph angiogenesis, sexual disorders, age-related macular degeneration, and treatment/ prevention of endometriosis and related conditions. Additional highly preferred indications include fibromas, heart disease, cardiac arrest, heart valve disease, and vascular disease. Preferred indications include blood disorders (e.g., as described below under “Immune Activity”, “Blood-Related Disorders”, and/or “Cardiovascular Disorders”). Preferred indications include autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis and/or as described below) and immunodeficiencies (e.g., as described below). Additional preferred indications include inflammation and inflammatory disorders (such as acute and chronic inflammatory diseases, e.g., inflammatory bowel disease and Crohn's disease), and pain management. 5 Activation of Kinase assay. JNK and p38 kinase assays A highly preferred embodiment of the invention Endothelial Cell p38 for signal transduction that regulate cell includes a method for stimulating endothelial or JNK Signaling proliferation, activation, or apoptosis are cell growth. An alternative highly preferred Pathway. well known in the art and may be used or embodiment of the invention includes a method routinely modified to assess the ability of for inhibiting endothelial cell growth. A polypeptides of the invention (including highly preferred embodiment of the invention antibodies and agonists or antagonists of the includes a method for stimulating endothelial invention) to promote or inhibit cell cell proliferation. An alternative highly proliferation, activation, and apoptosis. preferred embodiment of the invention includes Exemplary assays for JNK and p38 kinase a method for inhibiting endothelial cell activity that may be used or routinely proliferation. A highly preferred modified to test JNK and p38 kinase- embodiment of the invention includes a method induced activity of polypeptides of the for stimulating apoptosis of endothelial cells. invention (including antibodies and agonists An alternative highly preferred embodiment of or antagonists of the invention) include the the invention includes a method for inhibiting assays disclosed in Forrer et al., Biol Chem (e.g., decreasing) apoptosis of endothelial cells. 379(8-9): 1101-1110 (1998); Gupta et al., A highly preferred embodiment of the invention Exp Cell Res 247(2): 495-504 (1999); includes a method for stimulating (e.g., Kyriakis JM, Biochem Soc Symp 64: 29-48 increasing) endothelial cell activation. An (1999); Chang and Karin, Nature alternative highly preferred embodiment of the 410(6824): 37-40 (2001); and Cobb MH, invention includes a method for inhibiting (e.g., Prog Biophys Mol Biol 71(3-4): 479-500 decreasing) the activation of and/or inactivating (1999); the contents of each of which are endothelial cells. A highly preferred herein incorporated by reference in its embodiment of the invention includes a method entirety. Endothelial cells that may be used for stimulating angiogenisis. An alternative according to these assays are publicly highly preferred embodiment of the invention available (e.g., through the ATCC ™™). includes a method for inhibiting angiogenesis. Exemplary endothelial cells that may be A highly preferred embodiment of the invention used according to these assays include includes a method for reducing cardiac human umbilical vein endothelial cells hypertrophy. An alternative highly preferred (HUVEC), which are endothelial cells embodiment of the invention includes a method which line venous blood vessels, and are for inducing cardiac hypertrophy. Highly involved in functions that include, but are preferred indications include neoplastic diseases not limited to, angiogenesis, vascular (e.g., as described below under permeability, vascular tone, and immune “Hyperproliferative Disorders”), and disorders cell extravasation. of the cardiovascular system (e.g., heart disease, congestive heart failure, hypertension, aortic stenosis, cardiomyopathy, valvular regurgitation, left ventricular dysfunction, atherosclerosis and atherosclerotic vascular disease, diabetic nephropathy, intracardiac shunt, cardiac hypertrophy, myocardial infarction, chronic hemodynamic overload, and/or as described below under “Cardiovascular Disorders”). Highly preferred indications include cardiovascular, endothelial and/or angiogenic disorders (e.g., systemic disorders that affect vessels such as diabetes mellitus, as well as diseases of the vessels themselves, such as of the arteries, capillaries, veins and/or lymphatics). Highly preferred are indications that stimulate angiogenesis and/or cardiovascularization. Highly preferred are indications that inhibit angiogenesis and/or cardiovascularization. Highly preferred indications include antiangiogenic activity to treat solid tumors, leukemias, and Kaposi's sarcoma, and retinal disorders. Highly preferred indications include neoplasms and cancer, such as, Kaposi's sarcoma, hemangioma (capillary and cavernous), glomus tumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma, angiosarcoma, haemangiopericytoma, lymphangioma, lymphangiosarcoma. Highly preferred indications also include cancers such as, prostate, breast, lung, colon, pancreatic, esophageal, stomach, brain, liver, and urinary cancer. Preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Highly preferred indications also include arterial disease, such as, atherosclerosis, hypertension, coronary artery disease, inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenom, aneurysms, restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular disorders such as peripheral vascular disease, and cancer. Highly preferred indications also include trauma such as wounds, burns, and injured tissue (e.g., vascular injury such as, injury resulting from balloon angioplasty, and atheroschlerotic lesions), implant fixation, scarring, ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal diseases such as acute renal failure, and osteoporosis. Additional highly preferred indications include stroke, graft rejection, diabetic or other retinopathies, thrombotic and coagulative disorders, vascularitis, lymph angiogenesis, sexual disorders, age-related macular degeneration, and treatment/ prevention of endometriosis and related conditions. Additional highly preferred indications include fibromas, heart disease, cardiac arrest, heart valve disease, and vascular disease. Preferred indications include blood disorders (e.g., as described below under “Immune Activity”, “Blood-Related Disorders”, and/or ““Cardiovascular Disorders””). Preferred indications include autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis and/or as described below) and immunodeficiencies (e.g., as described below). Additional preferred indications include inflammation and inflammatory disorders (such as acute and chronic inflammatory diseases, e.g., inflammatory bowel disease and Crohn's disease), and pain management. 6 Activation of Kinase assay. Kinase assays, for example A highly preferred embodiment of the invention Hepatocyte ERK an Elk-1 kinase assay, for ERK signal includes a method for stimulating hepatocyte Signaling Pathway transduction that regulate cell proliferation cell proliferation. An alternative highly or differentiation are well known in the art preferred embodiment of the invention includes and may be used or routinely modified to a method for inhibiting hepatocyte cell assess the ability of polypeptides of the proliferation. A highly preferred invention (including antibodies and agonists embodiment of the invention includes a method or antagonists of the invention) to promote for stimulating hepatocyte cell differentiation. or inhibit cell proliferation, activation, and An alternative highly preferred embodiment of differentiation. Exemplary assays for ERK the invention includes a method for inhibiting kinase activity that may be used or routinely hepatocyte cell differentiation. A highly modified to test ERK kinase-induced preferred embodiment of the invention includes activity of polypeptides of the invention a method for activating hepatocyte cells. An (including antibodies and agonists or alternative highly preferred embodiment of the antagonists of the invention) include the invention includes a method for inhibiting the assays disclosed in Forrer et al., Biol Chem activation of and/or inactivating hepatocyte 379(8-9): 1101-1110 (1998); Kyriakis JM, cells. Highly preferred indications include Biochem Soc Symp 64: 29-48 (1999); disorders of the liver and/or endocrine disorders Chang and Karin, Nature 410(6824): 37-40 (e.g., as described below under “Endocrine (2001); and Cobb MH, Prog Biophys Mol Disorders”). Preferred indications include Biol 71(3-4): 479-500 (1999); the contents neoplastic diseases (e.g., as described below of each of which are herein incorporated by under “Hyperproliferative Disorders”), blood reference in its entirety. Rat liver hepatoma disorders (e.g., as described below under cells that may be used according to these “Immune Activity”, “Cardiovascular assays are publicly available (e.g., through Disorders”, and/or “Blood-Related Disorders”), the ATCC ™). Exemplary rat liver immune disorders (e.g., as described below hepatoma cells that may be used according under “Immune Activity”), neural disorders to these assays include H4lle cells, which (e.g., as described below under “Neural Activity are known to respond to glucocorticoids, and Neurological Diseases”), and infection insulin, or cAMP derivatives. (e.g., as described below under “Infectious Disease”). A highly preferred indication is diabetes mellitus. An additional highly preferred indication is a complication associated with diabetes (e.g., diabetic retinopathy, diabetic nephropathy, kidney disease (e.g., renal failure, nephropathy and/or other diseases and disorders as described in the “Renal Disorders” section below), diabetic neuropathy, nerve disease and nerve damage (e.g., due to diabetic neuropathy), blood vessel blockage, heart disease, stroke, impotence (e.g., due to diabetic neuropathy or blood vessel blockage), seizures, mental confusion, drowsiness, nonketotic hyperglycemic-hyperosmolar coma, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the “Cardiovascular Disorders” section below), dyslipidemia, endocrine disorders (as described in the “Endocrine Disorders” section below), neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, infection (e.g., infectious diseases and disorders as described in the “Infectious Diseases” section below, especially of the urinary tract and skin), carpal tunnel syndrome and Dupuytren's contracture). An additional highly preferred indication is obesity and/or complications associated with obesity. Additional highly preferred indications include weight loss or alternatively, weight gain. Additional highly preferred indications are complications associated with insulin resistance. Additonal highly preferred indications are disorders of the musculoskeletal systems including myopathies, muscular dystrophy, and/or as described herein. Additional highly preferred indications include, hepatitis, jaundice, gallstones, cirrhosis of the liver, degenerative or necrotic liver disease, alcoholic liver diseases, fibrosis, liver regeneration, metabolic disease, dyslipidemia and chlolesterol metabolism. Additional highly preferred indications include neoplasms and cancers, such as, hepatocarcinomas, other liver cancers, and colon and pancreatic cancer. Preferred indications also include prostate, breast, lung, esophageal, stomach, brain, and urinary cancer. Other preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. 7 Activation of JNK Kinase assay. JNK kinase assays for signal Highly preferred indications include asthma, Signaling Pathway in transduction that regulate cell proliferation, allergy, hypersensitivity reactions, immune cells (such activation, or apoptosis are well known in inflammation, and inflammatory disorders. as eosinophils). the art and may be used or routinely Additional highly preferred indications include modified to assess the ability of immune and hematopoietic disorders (e.g., as polypeptides of the invention (including described below under “Immune Activity”, and antibodies and agonists or antagonists of the “Blood-Related Disorders”), autoimmune invention) to promote or inhibit cell diseases (e.g., rheumatoid arthritis, systemic proliferation, activation, and apoptosis. lupus erythematosis, Crohn″s disease, multiple Exemplary assays for JNK kinase activity sclerosis and/or as described below), that may be used or routinely modified to immunodeficiencies (e.g., as described below). test JNK kinase-induced activity of Highly preferred indications also include polypeptides of the invention (including boosting or inhibiting immune cell antibodies and agonists or antagonists of the proliferation. Preferred indications include invention) include the assays disclosed in neoplastic diseases (e.g., leukemia, lymphoma, Forrer et al., Biol Chem 379(8-9): 1101-1110 and/or as described below under (1998); Gupta et al., Exp Cell Res “Hyperproliferative Disorders”). Highly 247(2): 495-504 (1999); Kyriakis JM, preferred indications include boosting an Biochem Soc Symp 64: 29-48 (1999); eosinophil-mediated immune response, and Chang and Karin, Nature 410(6824): 37-40 suppressing an eosinophil-mediated immune (2001); and Cobb MH, Prog Biophys Mol response. Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Exemplary cells that may be used according to these assays include eosinophils. Eosinophils are important in the late stage of allergic reactions; they are recruited to tissues and mediate the inflammatory response of late stage allergic reaction. Moreover, exemplary assays that may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate signal transduction, cell proliferation, activation, or apoptosis in eosinophils include assays disclosed and/or cited in: Zhang JP, et al., “Role of caspases in dexamethasone-induced apoptosis and activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase in human eosinophils” Clin Exp Immunol; Oct; 122(1): 20-7 (2000); Hebestreit H, et al., “Disruption of fas receptor signaling by nitric oxide in eosinophils” J Exp Med; Feb 2; 187(3): 415-25 (1998); J Allergy Clin Immunol 1999 Sep; 104(3 Pt 1): 565-74; and, Sousa AR, et al., “In vivo resistance to corticosteroids in bronchial asthma is associated with enhanced phosyphorylation of JUN N-terminal kinase and failure of prednisolone to inhibit JUN N-terminal kinase phosphorylation” J Allergy Clin Immunol; Sep; 104(3 Pt 1): 565-74 (1999); the contents of each of which are herein incorporated by reference in its entirety. 8 Activation of Natural Kinase assay. Kinase assays, for example A highly preferred embodiment of the invention Killer Cell ERK an Elk-1 kinase assay, for ERK signal includes a method for stimulating natural killer Signaling Pathway. transduction that regulate cell proliferation cell proliferation. An alternative highly or differentiation are well known in the art preferred embodiment of the invention includes and may be used or routinely modified to a method for inhibiting natural killer cell assess the ability of polypeptides of the proliferation. A highly preferred invention (including antibodies and agonists embodiment of the invention includes a method or antagonists of the invention) to promote for stimulating natural killer cell differentiation. or inhibit cell proliferation, activation, and An alternative highly preferred embodiment of differentiation. Exemplary assays for ERK the invention includes a method for inhibiting kinase activity that may be used or routinely natural killer cell differentiation. Highly modified to test ERK kinase-induced preferred indications include neoplastic diseases activity of polypeptides of the invention (e.g., as described below under (including antibodies and agonists or “Hyperproliferative Disorders”), blood antagonists of the invention) include the disorders (e.g., as described below under assays disclosed in Forrer et al., Biol Chem “Immune Activity”, “Cardiovascular 379(8-9): 1101-1110 (1998); Kyriakis JM, Disorders”, and/or “Blood-Related Disorders”), Biochem Soc Symp 64: 29-48 (1999); immune disorders (e.g., as described below Chang and Karin, Nature 410(6824): 37-40 under “Immune Activity”) and infections (e.g., (2001); and Cobb MH, Prog Biophys Mol as described below under “Infectious Disease”). Biol 71(3-4): 479-500 (1999); the contents Preferred indications include blood disorders of each of which are herein incorporated by (e.g., as described below under “Immune reference in its entirety. Natural killer cells Activity”, “Blood-Related Disorders”, and/or that may be used according to these assays “Cardiovascular Disorders”). Highly preferred are publicly available (e.g., through the indications include autoimmune diseases (e.g., ATCC ™). Exemplary natural killer cells rheumatoid arthritis, systemic lupus that may be used according to these assays erythematosis, multiple sclerosis and/or as include the human natural killer cell lines described below) and immunodeficiencies (e.g., (for example, NK-YT cells which have as described below). Additional highly cytolytic and cytotoxic activity) or primary preferred indications include inflammation and NK cells. inflammatory disorders. Highly preferred indications also include cancers such as, kidney, melanoma, prostate, breast, lung, colon, pancreatic, esophageal, stomach, brain, liver, urinary cancer, lymphoma and leukemias. Other preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Other highly preferred indications include, pancytopenia, leukopenia, leukemias, Hodgkin's disease, acute lymphocytic anemia (ALL), arthritis, asthma, AIDS, granulomatous disease, inflammatory bowel disease, sepsis, psoriasis, immune reactions to transplanted organs and tissues, endocarditis, meningitis, Lyme Disease, and allergies. 9 Activation of Skeletal Kinase assay. Kinase assays, for example A highly preferred embodiment of the invention Mucle Cell PI3 an GSK-3 kinase assay, for PI3 kinase includes a method for increasing muscle cell Kinase Signalling signal transduction that regulate glucose survival An alternative highly preferred Pathway metabolism and cell survivial are well- embodiment of the invention includes a method known in the art and may be used or for decreasing muscle cell survival. A routinely modified to assess the ability of preferred embodiment of the invention includes polypeptides of the invention (including a method for stimulating muscle cell antibodies and agonists or antagonists of the proliferation. In a specific embodiment, invention) to promote or inhibit glucose skeletal muscle cell proliferation is stimulated. metabolism and cell survival. Exemplary An alternative highly preferred embodiment of assays for PI3 kinase activity that may be the invention includes a method for inhibiting used or routinely modified to test PI3 muscle cell proliferation. In a specific kinase-induced activity of polypeptides of embodiment, skeletal muscle cell proliferation the invention (including antibodies and is inhibited. A preferred embodiment of the agonists or antagonists of the invention) invention includes a method for stimulating include assays disclosed in Forrer et al., muscle cell differentiation. In a specific Biol Chem 379(8-9): 1101-1110 (1998); embodiment, skeletal muscle cell differentiation Nikoulina et al., Diabetes 49(2): 263-271 is stimulated. An alternative highly preferred (2000); and Schreyer et al., Diabetes embodiment of the invention includes a method 48(8): 1662-1666 (1999), the contents of for inhibiting muscle cell differentiation. In a each of which are herein incorporated by specific embodiment, skeletal muscle cell reference in its entirety. Rat myoblast cells differentiation is inhibited. Highly preferred that may be used according to these assays indications include disorders of the are publicly available (e.g., through the musculoskeletal system. Preferred indications ATCC ™). Exemplary rat myoblast cells include neoplastic diseases (e.g., as described that may be used according to these assays below under “Hyperproliferative Disorders”), include L6 cells. L6 is an adherent rat endocrine disorders (e.g., as described below myoblast cell line, isolated from primary under ““Endocrine Disorders””), neural cultures of rat thigh muscle, that fuses to disorders (e.g., as described below under form multinucleated myotubes and striated ““Neural Activity and Neurological fibers after culture in differentiation media. Diseases””), blood disorders (e.g., as described below under “Immune Activity”, “Cardiovascular Disorders”, and/or “Blood- Related Disorders”), immune disorders (e.g., as described below under ““Immune Activity””), and infection (e.g., as described below under “Infectious Disease”). A highly preferred indication is diabetes mellitus. An additional highly preferred indication is a complication associated with diabetes (e.g., diabetic retinopathy, diabetic nephropathy, kidney disease (e.g., renal failure, nephropathy and/or other diseases and disorders as described in the ““Renal Disorders”” section below), diabetic neuropathy, nerve disease and nerve damage (e.g, due to diabetic neuropathy), blood vessel blockage, heart disease, stroke, impotence (e.g., due to diabetic neuropathy or blood vessel blockage), seizures, mental confusion, drowsiness, nonketotic hyperglycemic-hyperosmolar coma, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the ““Cardiovascular Disorders”” section below), dyslipidemia, endocrine disorders (as described in the ““Endocrine Disorders”” section below), neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, infections (e.g., infectious diseases and disorders as described in the ““Infectious Diseases”” section below, especially of the urinary tract and skin), carpal tunnel syndrome and Dupuytren's contracture). An additional highly preferred indication is obesity and/or complications associated with obesity. Additional highly preferred indications include weight loss or alternatively, weight gain. Additional highly preferred indications are complications associated with insulin resistance. Additonal highly preferred indications are disorders of the musculoskeletal system including myopathies, muscular dystrophy, and/or as described herein. Additional highly preferred indications include: myopathy, atrophy, congestive heart failure, cachexia, myxomas, fibromas, congenital cardiovascular abnormalities, heart disease, cardiac arrest, heart valve disease, and vascular disease. Highly preferred indications include neoplasms and cancer, such as, rhabdomyoma, rhabdosarcoma, stomach, esophageal, prostate, and urinary cancer. Preferred indications also include breast, lung, colon, pancreatic, brain, and liver cancer. Other preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, hyperplasia, metaplasia, and/or dysplasia. 10 Activation of Skeletal Kinase assay. Kinase assays, for example Muscle Cell ERK Elk-1 kinase assays, for ERK signal Signaling Pathway transduction that regulate cell proliferation or differentiation are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Le Marchand- Brustel Y, Exp Clin Endocrinol Diabetes 107(2): 126-132 (1999); Kyriakis JM, Biochem Soc Symp 64: 29-48 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 11 Activation of Skeletal Kinase assay. Kinase assays, for example Muscle Cell PI3 an GSK-3 kinase assay, for PI3 kinase Kinase Signaling signal transduction that regulate glucose Pathway metabolism and cell survival are well- known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to promote or inhibit glucose metabolism and cell survival. Exemplary assays for PI3 kinase activity that may be used or routinely modified to test PI3 kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Nikoulina et al., Diabetes 49(2): 263-271 (2000); and Schreyer et al., Diabetes 48(8): 1662-1666 (1999), the contents of each of which are herein incorporated by reference in its entirety. Rat myoblast cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary rat myoblast cells that may be used according to these assays include L6 cells. L6 is an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuses to form multinucleated myotubes and striated fibers after culture in differentiation media. 12 Activation of T-Cell Kinase assay. JNK and p38 kinase assays Preferred indications include neoplastic p38 or JNK Signaling for signal transduction that regulate cell diseases (e.g., as described below under Pathway. proliferation, activation, or apoptosis are “Hyperproliferative Disorders”), blood well known in the art and may be used or disorders (e.g., as described below under routinely modified to assess the ability of “Immune Activity”, “Cardiovascular polypeptides of the invention (including Disorders”, and/or “Blood-Related Disorders”), antibodies and agonists or antagonists of the and infection (e.g., an infectious disease as invention) to promote or inhibit immune described below under “Infectious Disease”). cell (e.g. T-cell) proliferation, activation, Highly preferred indications include and apoptosis. Exemplary assays for JNK autoimmune diseases (e.g., rheumatoid arthritis, and p38 kinase activity that may be used or systemic lupus erythematosis, multiple sclerosis routinely modified to test JNK and p38 and/or as described below) and kinase-induced activity of polypeptides of immunodeficiencies (e.g., as described below). the invention (including antibodies and Additional highly preferred indications include agonists or antagonists of the invention) inflammation and inflammatory disorders. include the assays disclosed in Forrer et al., Highly preferred indications also include Biol Chem 379(8-9): 1101-1110 (1998); neoplastic diseases (e.g., leukemia, lymphoma, Gupta et al., Exp Cell Res 247(2): 495-504 and/or as described below under (1999); Kyriakis JM, Biochem Soc Symp “Hyperproliferative Disorders”). Highly 64: 29-48 (1999); Chang and Karin, Nature preferred indications include neoplasms and 410(6824): 37-40 (2001); and Cobb MH, cancers, such as, leukemia, lymphoma, prostate, Prog Biophys Mol Biol 71(3-4): 479-500 breast, lung, colon, pancreatic, esophageal, (1999); the contents of each of which are stomach, brain, liver, and urinary cancer. Other herein incorporated by reference in its preferred indications include benign entirety. T cells that may be used according dysproliferative disorders and pre-neoplastic to these assays are publicly available (e.g., conditions, such as, for example, hyperplasia, through the ATCC ™). Exemplary mouse T metaplasia, and/or dysplasia. Preferred cells that may be used according to these indications include arthritis, asthma, AIDS, assays include the CTLL cell line, which is allergy, anemia, pancytopenia, leukopenia, an IL-2 dependent suspension-culture cell thrombocytopenia, Hodgkin″s disease, acute line with cytotoxic activity. lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt″s lymphoma, granulomatous disease, inflammatory bowel disease, sepsis, psoriasis, suppression of immune reactions to transplanted organs and tissues, endocarditis, meningitis, and Lyme Disease. 13 Activation of Assays for activation of transcription are Transcription well-known in the art and may be used and routinely modified to assess ability of polypeptides of the invention to inhibit or activate transcription. An example of such an assay follows: Cells were pretreated with SID supernatants or controls for 15-18 hours. SEAP activity was measured after 48 hours. LS174T is an epithelial colon adenocarcinoma cell line. Its tumourigenicity in nude mice make cell line LS174T a model for studies on the mechanism of synthesis and secretion of specific tumoral markers in colon cancer. See, Patan et al., Circ Res, 89(8): 732-39 (2001), the contents of which are herein incorporated by reference in its entirety. 14 Activation of Assays for the activation of transcription Preferred indications include neoplastic transcription through through the AP1 response element are diseases (e.g., as described below under AP1 response known in the art and may be used or “Hyperproliferative Disorders”), blood element in immune routinely modified to assess the ability of disorders (e.g., as described below under cells (such as T- polypeptides of the invention (including “Immune Activity”, “Cardiovascular cells). antibodies and agonists or antagonists of the Disorders”, and/or “Blood-Related Disorders”), invention) to modulate growth and other and infection (e.g., an infectious disease as cell functions. Exemplary assays for described below under “Infectious Disease”). transcription through the AP1 response Highly preferred indications include element that may be used or routinely autoimmune diseases (e.g., rheumatoid arthritis, modified to test AP1-response element systemic lupus erythematosis, multiple sclerosis activity of polypeptides of the invention and/or as described below) and (including antibodies and agonists or immunodeficiencies (e.g., as described below). antagonists of the invention) include assays Additional highly preferred indications include disclosed in Berger et al., Gene 66: 1-10 inflammation and inflammatory disorders. (1988); Cullen and Malm, Methods in Highly preferred indications also include Enzymol 216: 362-368 (1992); Henthorn et neoplastic diseases (e.g., leukemia, lymphoma, al., Proc Natl Acad Sci USA 85: 6342-6346 and/or as described below under (1988); Rellahan et al., J Biol Chem “Hyperproliferative Disorders”). Highly 272(49): 30806-30811 (1997); Chang et al., preferred indications include neoplasms and Mol Cell Biol 18(9): 4986-4993 (1998); and cancers, such as, leukemia, lymphoma, prostate, Fraser et al., Eur J Immunol 29(3): 838-844 breast, lung, colon, pancreatic, esophageal, (1999), the contents of each of which are stomach, brain, liver, and urinary cancer. Other herein incorporated by reference in its preferred indications include benign entirety. T cells that may be used according dysproliferative disorders and pre-neoplastic to these assays are publicly available (e.g., conditions, such as, for example, hyperplasia, through the ATCC ™). Exemplary mouse T metaplasia, and/or dysplasia. Preferred cells that may be used according to these indications include arthritis, asthma, AIDS, assays include the CTLL cell line, which is allergy, anemia, pancytopenia, leukopenia, an IL-2 dependent suspension-culture cell thrombocytopenia, Hodgkin's disease, acute line with cytotoxic activity. Additional lymphocytic anemia (ALL), plasmacytomas, exemplary mouse T cells that may be used multiple myeloma, Burkitt's lymphoma, according to these assays include the HT2 granulomatous disease, inflammatory bowel cell line, which is an IL-2 dependent disease, sepsis, psoriasis, suppression of suspension culture cell line that also immune reactions to transplanted organs and responds to IL-4. Exemplary human T cells tissues, endocarditis, meningitis, and Lyme that may be used according to these assays Disease. include the SUPT cell line, which is an IL-2 and IL-4 responsive suspension-culture cell line. 15 Activation of Assays for the activation of transcription A highly preferred indication is obesity and/or transcription through through the cAMP response element are complications associated with obesity. cAMP response well-known in the art and may be used or Additional highly preferred indications include element (CRE) in routinely modified to assess the ability of weight loss or alternatively, weight gain. An pre-adipocytes. polypeptides of the invention (including additional highly preferred indication is antibodies and agonists or antagonists of the diabetes mellitus. An additional highly invention) to increase cAMP, regulate preferred indication is a complication CREB transcription factors, and modulate associated with diabetes (e.g., diabetic expression of genes involved in a wide retinopathy, diabetic nephropathy, kidney variety of cell functions. For example, a disease (e.g., renal failure, nephropathy and/or 3T3-L1/CRE reporter assay may be used to other diseases and disorders as described in the identify factors that activate the cAMP “Renal Disorders” section below), diabetic signaling pathway. CREB plays a major neuropathy, nerve disease and nerve damage role in adipogenesis, and is involved in (e.g., due to diabetic neuropathy), blood vessel differentiation into adipocytes. CRE blockage, heart disease, stroke, impotence (e.g., contains the binding sequence for the due to diabetic neuropathy or blood vessel transcription factor CREB (CRE binding blockage), seizures, mental confusion, protein). Exemplary assays for transcription drowsiness, nonketotic hyperglycemic- through the cAMP response element that hyperosmolar coma, cardiovascular disease may be used or routinely modified to test (e.g., heart disease, atherosclerosis, cAMP-response element activity of microvascular disease, hypertension, stroke, polypeptides of the invention (including and other diseases and disorders as described in antibodies and agonists or antagonists of the the “Cardiovascular Disorders” section below), invention) include assays disclosed in dyslipidemia, endocrine disorders (as described Berger et al., Gene 66: 1-10 (1998); Cullen in the “Endocrine Disorders” section below), and Malm, Methods in Enzymol 216: 362-368 neuropathy, vision impairment (e.g., diabetic (1992); Henthorn et al., Proc Natl Acad retinopathy and blindness), ulcers and impaired Sci USA 85: 6342-6346 (1988); Reusch et wound healing, and infection (e.g., infectious al., Mol Cell Biol 20(3): 1008-1020 (2000); diseases and disorders as described in the and Klemm et al., J Biol Chem 273: 917-923 “Infectious Diseases” section below, especially (1998), the contents of each of which are of the urinary tract and skin), carpal tunnel herein incorporated by reference in its syndrome and Dupuytren's contracture). entirety. Pre-adipocytes that may be used Additional highly preferred indications are according to these assays are publicly complications associated with insulin available (e.g., through the ATCC ™) and/or resistance. may be routinely generated. Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3-L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre- adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. 16 Activation of Assays for the activation of transcription Preferred indications include blood disorders transcription through through the cAMP response element are (e.g., as described below under “Immune cAMP response well-known in the art and may be used or Activity”, “Blood-Related Disorders”, and/or element in immune routinely modified to assess the ability of “Cardiovascular Disorders”), and infection cells (such as T- polypeptides of the invention (including (e.g., an infectious disease as described below cells). antibodies and agonists or antagonists of the under “Infectious Disease”). Preferred invention) to increase cAMP, bind to CREB indications include autoimmune diseases (e.g., transcription factor, and modulate rheumatoid arthritis, systemic lupus expression of genes involved in a wide erythematosis, multiple sclerosis and/or as variety of cell functions. Exemplary assays described below), immunodeficiencies (e.g., as for transcription through the cAMP described below), boosting a T cell-mediated response element that may be used or immune response, and suppressing a T cell- routinely modified to test cAMP-response mediated immune response. Additional element activity of polypeptides of the preferred indications include inflammation and invention (including antibodies and agonists inflammatory disorders. Highly preferred or antagonists of the invention) include indications include neoplastic diseases (e.g., assays disclosed in Berger et al., Gene 66: 1-10 leukemia, lymphoma, and/or as described (1998); Cullen and Malm, Methods in below under “Hyperproliferative Disorders”). Enzymol 216: 362-368 (1992); Henthorn et Highly preferred indications include neoplasms al., Proc Natl Acad Sci USA 85: 6342-6346 and cancers, such as, leukemia, lymphoma (e.g., (1988); Black et al., Virus Genes 15(2): 105-117 T cell lymphoma, Burkitt's lymphoma, non- (1997); and Belkowski et al., J Hodgkins lymphoma, Hodgkin″s disease), Immunol 161(2): 659-665 (1998), the melanoma, and prostate, breast, lung, colon, contents of each of which are herein pancreatic, esophageal, stomach, brain, liver incorporated by reference in its entirety. T and urinary cancer. Other preferred indications cells that may be used according to these include benign dysproliferative disorders and assays are publicly available (e.g., through pre-neoplastic conditions, such as, for example, the ATCC ™). Exemplary human T cells hyperplasia, metaplasia, and/or dysplasia. that may be used according to these assays Preferred indications include anemia, include the JURKAT cell line, which is a pancytopenia, leukopenia, thrombocytopenia, suspension culture of leukemia cells that acute lymphocytic anemia (ALL), produce IL-2 when stimulated. Exemplary plasmacytomas, multiple myeloma, arthritis, mouse T cells that may be used according to AIDS, granulomatous disease, inflammatory these assays include the CTLL cell line, bowel disease, sepsis, neutropenia, neutrophilia, which is a suspension culture of IL-2 psoriasis, suppression of immune reactions to dependent cytotoxic T cells. Aditional transplanted organs and tissues, hemophilia, exemplary mouse T cells that may be used hypercoagulation, diabetes mellitus, according to these assays include the HT2 endocarditis, meningitis, Lyme Disease, and cell line, which is a suspension culture of asthma and allergy. IL-2 dependent T cells that also respond to IL-4. 17 Activation of Assays for the activation of transcription A highly preferred embodiment of the invention transcription through through the CD28 response element are includes a method for stimulating T cell CD28 response well-known in the art and may be used or proliferation. An alternative highly preferred element in immune routinely modified to assess the ability of embodiment of the invention includes a method cells (such as T- polypeptides of the invention (including for inhibiting T cell proliferation. A highly cells). antibodies and agonists or antagonists of the preferred embodiment of the invention includes invention) to stimulate IL-2 expression in T a method for activating T cells. An alternative cells. Exemplary assays for transcription highly preferred embodiment of the invention through the CD28 response element that includes a method for inhibiting the activation may be used or routinely modified to test of and/or inactivating T cells. A highly CD28-response element activity of preferred embodiment of the invention includes polypeptides of the invention (including a method for stimulating (e.g., increasing) IL-2 antibodies and agonists or antagonists of the production. An alternative highly preferred invention) include assays disclosed in embodiment of the invention includes a method Berger et al., Gene 66: 1-10 (1998); Cullen for inhibiting (e.g., reducing) IL-2 production. and Malm, Methods in Enzymol 216: 362-368 Additional highly preferred indications include (1992); Henthorn et al., Proc Natl Acad inflammation and inflammatory disorders. Sci USA 85: 6342-6346 (1988); McGuire Highly preferred indications include and Iacobelli, J Immunol 159(3): 1319-1327 autoimmune diseases (e.g., rheumatoid arthritis, (1997); Parra et al., J Immunol 166(4): 2437-2443 systemic lupus erythematosis, multiple sclerosis (2001); and Butscher et al., J Biol and/or as described below), Chem 3(1): 552-560 (1998), the contents of immunodeficiencies (e.g., as described below), each of which are herein incorporated by boosting a T cell-mediated immune response, reference in its entirety. T cells that may be and suppressing a T cell-mediated immune used according to these assays are publicly response. Highly preferred indications available (e.g., through the ATCC ™). include neoplastic diseases (e.g., melanoma, Exemplary human T cells that may be used renal cell carcinoma, leukemia, lymphoma, according to these assays include the SUPT and/or as described below under cell line, which is a suspension culture of “Hyperproliferative Disorders”). Highly IL-2 and IL-4 responsive T cells. Additional preferred indications include neoplasms and exemplary human T cells that may be used cancers, such as, for example, melanoma (e.g., according to these assays include the metastatic melanoma), renal cell carcinoma JURKAT cell line, which is a suspension (e.g., metastatic renal cell carcinoma), culture of leukemia cells that produce IL-2 leukemia, lymphoma (e.g,. T cell lymphoma), when stimulated. and prostate, breast, lung, colon, pancreatic, esophageal, stomach, brain, liver and urinary cancer. Other preferred indications include benign dysproliferative disorders and pre- neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. A highly preferred indication includes infection (e.g., AIDS, tuberculosis, infections associated with granulomatous disease, and osteoporosis, and/or as described below under “Infectious Disease”). A highly preferred indication is AIDS. Additional highly preferred indications include suppression of immune reactions to transplanted organs and/or tissues, uveitis, psoriasis, and tropical spastic paraparesis. Preferred indications include blood disorders (e.g., as described below under “Immune Activity”, “Blood-Related Disorders”, and/or “Cardiovascular Disorders”). Preferred indications also include anemia, pancytopenia, leukopenia, thrombocytopenia, Hodgkin's disease, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, granulomatous disease, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, asthma and allergy. 18 Activation of Assays for the activation of transcription A highly preferred embodiment of the invention transcription through through the Gamma Interferon Activation includes a method for stimulating endothelial GAS response Site (GAS) response element are well- cell growth. An alternative highly preferred element in epithelial known in the art and may be used or embodiment of the invention includes a method cells (such as HELA routinely modified to assess the ability of for inhibiting endothelial cell growth. A cells). polypeptides of the invention (including highly preferred embodiment of the invention antibodies and agonists or antagonists of the includes a method for stimulating endothelial invention) to regulate STAT transcription cell proliferation. An alternative highly factors and modulate gene expression preferred embodiment of the invention includes involved in a wide variety of cell functions. a method for inhibiting endothelial cell Exemplary assays for transcription through proliferation. A highly preferred the GAS response element that may be used embodiment of the invention includes a method or routinely modified to test GAS-response for stimulating apoptosis of endothelial cells. element activity of polypeptides of the An alternative highly preferred embodiment of invention (including antibodies and agonists the invention includes a method for inhibiting or antagonists of the invention) include (e.g., decreasing) apoptosis of endothelial cells. assays disclosed in: You M, et al, J Biol A highly preferred embodiment of the invention Chem, 272(37): 23376-23381(1997); Min W, includes a method for stimulating (e.g., et al., Circ Res, 83(8): 815-823 (1998); increasing) endothelial cell activation. An Berger et al., Gene 66: 1-10 (1998); Cullen alternative highly preferred embodiment of the and Malm, Methods in Enzymol 216: 362-368 invention includes a method for inhibiting (e.g., (1992); Henthorn et al., Proc Natl Acad decreasing) the activation of and/or inactivating Sci USA 85: 6342-6346 (1988); Matikainen endothelial cells. A highly preferred et al., Blood 93(6): 1980-1991 (1999); and embodiment of the invention includes a method Henttinen et al., J Immunol 155(10): 4582-4587 for stimulating angiogenisis. An alternative (1995), the contents of each of which highly preferred embodiment of the invention are herein incorporated by reference in its includes a method for inhibiting angiogenesis. entirety. Epithelial cells that may be used A highly preferred embodiment of the invention according to these assays are publicly includes a method for reducing cardiac available (e.g., through the ATCC ™). hypertrophy. An alternative highly preferred Exemplary epithelial cells that may be used embodiment of the invention includes a method according to these assays include the HELA for inducing cardiac hypertrophy. Preferred cell line. embodiments of the invention include using polypeptides of the invention (or antibodies, agonists, or antagonists thereof) in detection, diagnosis, prevention, and/or treatment of Cancer, Wound Healing, and Inflamation. Highly preferred indications include neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., heart disease, congestive heart failure, hypertension, aortic stenosis, cardiomyopathy, valvular regurgitation, left ventricular dysfunction, atherosclerosis and atherosclerotic vascular disease, diabetic nephropathy, intracardiac shunt, cardiac hypertrophy, myocardial infarction, chronic hemodynamic overload, and/or as described below under “Cardiovascular Disorders”). Highly preferred indications include cardiovascular, endothelial and/or angiogenic disorders (e.g., systemic disorders that affect vessels such as diabetes mellitus, as well as diseases of the vessels themselves, such as of the arteries, capillaries, veins and/or lymphatics). Highly preferred are indications that stimulate angiogenesis and/or cardiovascularization. Highly preferred are indications that inhibit angiogenesis and/or cardiovascularization. Highly preferred indications include antiangiogenic activity to treat solid tumors, leukemias, and Kaposi's sarcoma, and retinal disorders. Highly preferred indications include neoplasms and cancers, such as, Kaposi's sarcoma, hemangioma (capillary and cavernous), glomus tumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma, angiosarcoma, haemangiopericytoma, lymphangioma, lymphangiosarcoma. Highly preferred indications also include cancers such as, melanoma and prostate, breast, lung, colon, pancreatic, esophageal, stomach, brain, liver, and urinary cancer. Other preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Highly preferred indications also include arterial disease, such as, atherosclerosis, hypertension, coronary artery disease, inflammatory vasculitides, Reynaud's disease and Reynaud's phenomenom, aneurysms, restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular disorders such as peripheral vascular disease, and cancer. Highly preferred indications also include trauma such as wounds, burns, and injured tissue (e.g., vascular injury such as, injury resulting from balloon angioplasty, and atheroschlerotic lesions), implant fixation, scarring, ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal diseases such as acute renal failure, and osteoporosis. Additional highly preferred indications include stroke, graft rejection, diabetic or other retinopathies, thrombotic and coagulative disorders, vascularitis, lymph angiogenesis, sexual disorders, age-related macular degeneration, and treatment/ prevention of endometriosis and related conditions. Additional highly preferred indications include fibromas, heart disease, cardiac arrest, heart valve disease, and vascular disease. Preferred indications include blood disorders (e.g., as described below under “Immune Activity”, “Blood-Related Disorders”, and/or ““Cardiovascular Disorders””). Preferred indications include autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis and/or as described below) and immunodeficiencies (e.g., as described below). Additional preferred indications include inflammation and inflammatory disorders (such as acute and chronic inflammatory diseases, e.g., inflammatory bowel disease and Crohn's disease), and pain management. 19 Activation of Assays for the activation of transcription Highly preferred indications include asthma, transcription through through the Gamma Interferon Activation allergy, hypersensitivity reactions, GAS response Site (GAS) response element are well- inflammation, and inflammatory disorders. element in immune known in the art and may be used or Additional highly preferred indications include cells (such as routinely modified to assess the ability of immune and hematopoietic disorders (e.g., as eosinophils). polypeptides of the invention (including described below under “Immune Activity”, and antibodies and agonists or antagonists of the “Blood-Related Disorders”), autoimmune invention) to modulate gene expression diseases (e.g., rheumatoid arthritis, systemic (commonly via STAT transcription factors) lupus erythematosis, Crohn″s disease, multiple involved in a wide variety of cell functions. sclerosis and/or as described below), Exemplary assays for transcription through immunodeficiencies (e.g., as described below), the GAS response element that may be used boosting an eosinophil-mediated immune or routinely modified to test GAS-response response and, alternatively, suppressing an element activity of polypeptides of the eosinophil-mediated immune response. invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995); the contents of each of which are herein incorporated by reference in its entirety. Moreover, exemplary assays that may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to activate or inhibit activation of immune cells include assays disclosed and/or cited in: Mayumi M., “EoL-1, a human eosinophilic cell line” Leuk Lymphoma; Jun; 7(3): 243-50 (1992); Bhattacharya S, “Granulocyte macrophage colony-stimulating factor and interleukin-5 activate STAT5 and induce CIS1 mRNA in human peripheral blood eosinophils” Am J Respir Cell Mol Biol; Mar; 24(3): 312-6 (2001); and, Du J, et al., “Engagement of the CrkL adapter in interleukin-5 signaling in eosinophils” J Biol Chem; Oct 20; 275(42): 33167-75 (2000); the contents of each of which are herein incorporated by reference in its entirety. Exemplary cells that may be used according to these assays include eosinophils. Eosinophils are a type of immune cell important in the late stage of allergic reactions; they are recruited to tissues and mediate the inflammatory response of late stage allergic reaction. Increases in GAS mediated transcription in eosinophils is typically a result of STAT activation, normally a direct consequence of interleukin or other cytokine receptor stimulation (e.g. IL3, IL5 or GMCSF). 20 Activation of Assays for the activation of transcription Preferred embodiments of the invention include transcription through through the Gamma Interferon Activation using polypeptides of the invention (or GAS response Site (GAS) response element are well- antibodies, agonists, or antagonists thereof) in element in immune known in the art and may be used or detection, diagnosis, prevention, and/or cells (such as routinely modified to assess the ability of treatment of Inflammation, Infection, Cancer, monocytes). polypeptides of the invention (including Hypersensitivity, and Atherosclerosis. antibodies and agonists or antagonists of the invention) to regulate STAT transcription factors and modulate gene expression involved in a wide variety of cell functions. Exemplary assays for transcription through the GAS response element that may be used or routinely modified to test GAS-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Gustafson KS, et al., J Biol Chem, 271(33): 20035-20046 (1996); Eilers A, et al., Immunobiology, 193(2-4): 328-333 (1995); Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Matikainen et al., Blood 93(6): 1980-1991 (1999); and Henttinen et al., J Immunol 155(10): 4582-4587 (1995), the contents of each of which are herein incorporated by reference in its entirety. Exemplary immune cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary immune cells that may be used according to these assays include the U937 cell line, which is a monocytic cell line. 21 Activation of Assays for the activation of transcription Highly preferred indications include neoplastic transcription through through the Gamma Interferon Activation diseases (e.g., leukemia, lymphoma, and/or as GAS response Site (GAS) response element are well- described below under “Hyperproliferative element in immune known in the art and may be used or Disorders”). Highly preferred indications cells (such as T- routinely modified to assess the ability of include neoplasms and cancers, such as, for cells). polypeptides of the invention (including example, leukemia, lymphoma (e.g., T cell antibodies and agonists or antagonists of the lymphoma, Burkitt's lymphoma, non-Hodgkins invention) to regulate STAT transcription lymphoma, Hodgkin's disease), melanoma, and factors and modulate gene expression prostate, breast, lung, colon, pancreatic, involved in a wide variety of cell functions. esophageal, stomach, brain, liver and urinary Exemplary assays for transcription through cancer. Other preferred indications include the GAS response element that may be used benign dysproliferative disorders and pre- or routinely modified to test GAS-response neoplastic conditions, such as, for example, element activity of polypeptides of the hyperplasia, metaplasia, and/or dysplasia. invention (including antibodies and agonists Preferred indications include autoimmune or antagonists of the invention) include diseases (e.g., rheumatoid arthritis, systemic assays disclosed in Berger et al., Gene 66: 1-10 lupus erythematosis, multiple sclerosis and/or (1998); Cullen and Malm, Methods in as described below), immunodeficiencies (e.g., Enzymol 216: 362-368 (1992); Henthorn et as described below), boosting a T cell-mediated al., Proc Natl Acad Sci USA 85: 6342-6346 immune response, and suppressing a T cell- (1988); Matikainen et al., Blood mediated immune response. Additional 93(6): 1980-1991 (1999); and Henttinen et preferred indications include inflammation and al., J Immunol 155(10): 4582-4587 (1995), inflammatory disorders. Highly preferred the contents of each of which are herein indications include blood disorders (e.g., as incorporated by reference in its entirety. described below under “Immune Activity”, Exemplary mouse T cells that may be used “Blood-Related Disorders”, and/or according to these assays are publicly ““Cardiovascular Disorders””), and infection available (e.g., through the ATCC ™). (e.g., viral infections, tuberculosis, infections Exemplary human T cells, such as the associated with chronic granulomatosus disease MOLT4 cell line, that may be used and malignant osteoporosis, and/or an according to these assays are publicly infectious disease as described below under available (e.g., through the ATCC ™). “Infectious Disease”). An additional preferred Additional exemplary human T cells, such indication is idiopathic pulmonary fibrosis. as the SUPT cell line, that may be used Preferred indications include anemia, according to these assays are publicly pancytopenia, leukopenia, thrombocytopenia, available (e.g., through the ATCC ™). acute lymphocytic anemia (ALL), Additional exemplary T cells that may be plasmacytomas, multiple myeloma, arthritis, used according to these assays include the AIDS, granulomatous disease, inflammatory CTLL cell line, which is a suspension bowel disease, sepsis, neutropenia, neutrophilia, culture of IL-2 dependent cytotoxic T cells. psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and asthma and allergy. 22 Activation of This reporter assay measures activation of Highly preferred indications include allergy, transcription through the GATA-3 signaling pathway in HMC-1 asthma, and rhinitis. Additional preferred GATA-3 response human mast cell line. Activation of GATA- indications include infection (e.g., an infectious element in immune 3 in mast cells has been linked to cytokine disease as described below under “Infectious cells (such as mast and chemokine production. Assays for the Disease”), and inflammation and inflammatory cells). activation of transcription through the disorders. Preferred indications also include GATA3 response element are well-known blood disorders (e.g., as described below under in the art and may be used or routinely “Immune Activity”, “Blood-Related modified to assess the ability of Disorders”, and/or “Cardiovascular Disorders”). polypeptides of the invention (including Preferred indications include autoimmune antibodies and agonists or antagonists of the diseases (e.g., rheumatoid arthritis, systemic invention) to regulate GATA3 transcription lupus erythematosis, multiple sclerosis and/or factors and modulate expression of mast cell as described below) and immunodeficiencies genes important for immune response (e.g., as described below). Preferred indications development. Exemplary assays for include neoplastic diseases (e.g., leukemia, transcription through the GATA3 response lymphoma, melanoma, prostate, breast, lung, element that may be used or routinely colon, pancreatic, esophageal, stomach, brain, modified to test GATA3-response element liver, and urinary tract cancers and/or as activity of polypeptides of the invention described below under “Hyperproliferative (including antibodies and agonists or Disorders”). Other preferred indications antagonists of the invention) include assays include benign dysproliferative disorders and disclosed in Berger et al., Gene 66: 1-10 pre-neoplastic conditions, such as, for example, (1998); Cullen and Malm, Methods in hyperplasia, metaplasia, and/or dysplasia. Enzymol 216: 362-368 (1992); Henthorn et Preferred indications include anemia, al., Proc Natl Acad Sci USA 85: 6342-6346 pancytopenia, leukopenia, thrombocytopenia, (1988); Flavell et al., Cold Spring Harb leukemias, Hodgkin's disease, acute Symp Quant Biol 64: 563-571 (1999); lymphocytic anemia (ALL), plasmacytomas, Rodriguez-Palmero et al., Eur J Immunol multiple myeloma, Burkitt's lymphoma, 29(12): 3914-3924 (1999); Zheng and arthritis, AIDS, granulomatous disease, Flavell, Cell 89(4): 587-596 (1997); and inflammatory bowel disease, sepsis, Henderson et al., Mol Cell Biol 14(6): 4286-4294 neutropenia, neutrophilia, psoriasis, suppression (1994), the contents of each of which of immune reactions to transplanted organs and are herein incorporated by reference in its tissues, hemophilia, hypercoagulation, diabetes entirety. Mast cells that may be used mellitus, endocarditis, meningitis, and Lyme according to these assays are publicly Disease. available (e.g., through the ATCC ™). Exemplary human mast cells that may be used according to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 23 Activation of Assays for the activation of transcription transcription through through the GATA3 response element are GATA-3 response well-known in the art and may be used or element in immune routinely modified to assess the ability of cells (such as T- polypeptides of the invention (including cells). antibodies and agonists or antagonists of the invention) to regulate GATA3 transcription factors and modulate expression of genes important for Th2 immune response development. Exemplary assays for transcription through the GATA3 response element that may be used or routinely modified to test GATA3-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Flavell et al., Cold Spring Harb Symp Quant Biol 64: 563-571 (1999); Rodriguez-Palmero et al., Eur J Immunol 29(12): 3914-3924 (1999); Zheng and Flavell, Cell 89(4): 587-596 (1997); and Henderson et al., Mol Cell Biol 14(6): 4286-4294 (1994), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary mouse T cells that may be used according to these assays include the HT2 cell line, which is a suspension culture of IL-2 dependent T cells that also respond to IL-4. 24 Activation of This reporter assay measures activation of Highly preferred indications include allergy, transcription through the NFAT signaling pathway in HMC-1 asthma, and rhinitis. Additional preferred NFAT response human mast cell line. Activation of NFAT indications include infection (e.g., an infectious element in immune in mast cells has been linked to cytokine disease as described below under “Infectious cells (such as mast and chemokine production. Assays for the Disease”), and inflammation and inflammatory cells). activation of transcription through the disorders. Preferred indications also include Nuclear Factor of Activated T cells (NFAT) blood disorders (e.g., as described below under response element are well-known in the art “Immune Activity”, “Blood-Related and may be used or routinely modified to Disorders”, and/or “Cardiovascular Disorders”). assess the ability of polypeptides of the Preferred indications include autoimmune invention (including antibodies and agonists diseases (e.g., rheumatoid arthritis, systemic or antagonists of the invention) to regulate lupus erythematosis, multiple sclerosis and/or NFAT transcription factors and modulate as described below) and immunodeficiencies expression of genes involved in (e.g., as described below). Preferred indications immunomodulatory functions. Exemplary include neoplastic diseases (e.g., leukemia, assays for transcription through the NFAT lymphoma, melanoma, prostate, breast, lung, response element that may be used or colon, pancreatic, esophageal, stomach, brain, routinely modified to test NFAT-response liver, and urinary tract cancers and/or as element activity of polypeptides of the described below under “Hyperproliferative invention (including antibodies and agonists Disorders”). Other preferred indications or antagonists of the invention) include include benign dysproliferative disorders and assays disclosed in Berger et al., Gene 66: 1-10 pre-neoplastic conditions, such as, for example, (1998); Cullen and Malm, Methods in hyperplasia, metaplasia, and/or dysplasia. Enzymol 216: 362-368 (1992); Henthorn et Preferred indications include anemia, al., Proc Natl Acad Sci USA 85: 6342-6346 pancytopenia, leukopenia, thrombocytopenia, (1988); De Boer et al., Int J Biochem Cell leukemias, Hodgkin's disease, acute Biol 31(10): 1221-1236 (1999); Ali et al., J lymphocytic anemia (ALL), plasmacytomas, Immunol 165(12): 7215-7223 (2000); multiple myeloma, Burkitt's lymphoma, Hutchinson and McCloskey, J Biol Chem arthritis, AIDS, granulomatous disease, 270(27): 16333-16338 (1995), and Turner et inflammatory bowel disease, sepsis, al., J Exp Med 188: 527-537 (1998), the neutropenia, neutrophilia, psoriasis, suppression contents of each of which are herein of immune reactions to transplanted organs and incorporated by reference in its entirety. tissues, hemophilia, hypercoagulation, diabetes Mast cells that may be used according to mellitus, endocarditis, meningitis, and Lyme these assays are publicly available (e.g., Disease. through the ATCC ™). Exemplary human mast cells that may be used according to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 25 Activation of Assays for the activation of transcription Highly preferred indications include blood transcription through through the Nuclear Factor of Activated T disorders (e.g., as described below under NFAT response cells (NFAT) response element are well- “Immune Activity”, “Blood-Related element in immune known in the art and may be used or Disorders”, and/or “Cardiovascular Disorders”). cells (such as natural routinely modified to assess the ability of Highly preferred indications include killer cells). polypeptides of the invention (including autoimmune diseases (e.g., rheumatoid arthritis, antibodies and agonists or antagonists of the systemic lupus erythematosis, multiple sclerosis invention) to regulate NFAT transcription and/or as described below), factors and modulate expression of genes immunodeficiencies (e.g., as described below), involved in immunomodulatory functions. boosting a T cell-mediated immune response, Exemplary assays for transcription through and suppressing a T cell-mediated immune the NFAT response element that may be response. Additional highly preferred used or routinely modified to test NFAT- indications include inflammation and response element activity of polypeptides of inflammatory disorders. An additional highly the invention (including antibodies and preferred indication is infection (e.g., an agonists or antagonists of the invention) infectious disease as described below under include assays disclosed in Berger et al., “Infectious Disease”). Preferred indications Gene 66: 1-10 (1998); Cullen and Malm, include neoplastic diseases (e.g., leukemia, Methods in Enzymol 216: 362-368 (1992); lymphoma, and/or as described below under Henthorn et al., Proc Natl Acad Sci USA “Hyperproliferative Disorders”). Preferred 85: 6342-6346 (1988); Aramburu et al., J indications include neoplasms and cancers, such Exp Med 182(3): 801-810 (1995); De Boer as, for example, leukemia, lymphoma, and et al., Int J Biochem Cell Biol 31(10): 1221-1236 prostate, breast, lung, colon, pancreatic, (1999); Fraser et al., Eur J Immunol esophageal, stomach, brain, liver and urinary 29(3): 838-844 (1999); and Yeseen et al., J cancer. Other preferred indications include Biol Chem 268(19): 14285-14293 (1993), benign dysproliferative disorders and pre- the contents of each of which are herein neoplastic conditions, such as, for example, incorporated by reference in its entirety. hyperplasia, metaplasia, and/or dysplasia. NK cells that may be used according to Preferred indications also include anemia, these assays are publicly available (e.g., pancytopenia, leukopenia, thrombocytopenia, through the ATCC ™). Exemplary human Hodgkin's disease, acute lymphocytic anemia NK cells that may be used according to (ALL), plasmacytomas, multiple myeloma, these assays include the NK-YT cell line, Burkitt's lymphoma, arthritis, AIDS, which is a human natural killer cell line granulomatous disease, inflammatory bowel with cytolytic and cytotoxic activity. disease, sepsis, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, asthma and allergy. 26 Activation of Assays for the activation of transcription Highly preferred indications include blood transcription through through the Nuclear Factor of Activated T disorders (e.g., as described below under NFAT response cells (NFAT) response element are well- “Immune Activity”, “Blood-Related element in immune known in the art and may be used or Disorders”, and/or “Cardiovascular Disorders”). cells (such as T- routinely modified to assess the ability of Highly preferred indications include cells). polypeptides of the invention (including autoimmune diseases (e.g., rheumatoid arthritis, antibodies and agonists or antagonists of the systemic lupus erythematosis, multiple sclerosis invention) to regulate NFAT transcription and/or as described below), factors and modulate expression of genes immunodeficiencies (e.g., as described below), involved in immunomodulatory functions. boosting a T cell-mediated immune response, Exemplary assays for transcription through and suppressing a T cell-mediated immune the NFAT response element that may be response. Additional highly preferred used or routinely modified to test NFAT- indications include inflammation and response element activity of polypeptides of inflammatory disorders. An additional highly the invention (including antibodies and preferred indication is infection (e.g., an agonists or antagonists of the invention) infectious disease as described below under include assays disclosed in Berger et al., “Infectious Disease”). Preferred indications Gene 66: 1-10 (1998); Cullen and Malm, include neoplastic diseases (e.g., leukemia, Methods in Enzymol 216: 362-368 (1992); lymphoma, and/or as described below under Henthorn et al., Proc Natl Acad Sci USA “Hyperproliferative Disorders”). Preferred 85: 6342-6346 (1988); Serfling et al., indications include neoplasms and cancers, such Biochim Biophys Acta 1498(1): 1-18 as, for example, leukemia, lymphoma, and (2000); De Boer et al., Int J Biochem Cell prostate, breast, lung, colon, pancreatic, Biol 31(10): 1221-1236 (1999); Fraser et al., esophageal, stomach, brain, liver and urinary Eur J Immunol 29(3): 838-844 (1999); and cancer. Other preferred indications include Yeseen et al., J Biol Chem 268(19): 14285-14293 benign dysproliferative disorders and pre- (1993), the contents of each of which neoplastic conditions, such as, for example, are herein incorporated by reference in its hyperplasia, metaplasia, and/or dysplasia. entirety. T cells that may be used according Preferred indications also include anemia, to these assays are publicly available (e.g., pancytopenia, leukopenia, thrombocytopenia, through the ATCC ™). Exemplary human T Hodgkin's disease, acute lymphocytic anemia cells that may be used according to these (ALL), plasmacytomas, multiple myeloma, assays include the SUPT cell line, which is Burkitt's lymphoma, arthritis, AIDS, a suspension culture of IL-2 and IL-4 granulomatous disease, inflammatory bowel responsive T cells. disease, sepsis, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, asthma and allergy. 27 Activation of Assays for the activation of transcription Highly preferred indications include blood transcription through through the Nuclear Factor of Activated T disorders (e.g., as described below under NFAT response in cells (NFAT) response element are well- “Immune Activity”, “Blood-Related immune cells (such known in the art and may be used or Disorders”, and/or “Cardiovascular Disorders”). as T-cells). routinely modified to assess the ability of Highly preferred indications include polypeptides of the invention (including autoimmune diseases (e.g., rheumatoid arthritis, antibodies and agonists or antagonists of the systemic lupus erythematosis, multiple sclerosis invention) to regulate NFAT transcription and/or as described below), factors and modulate expression of genes immunodeficiencies (e.g., as described below), involved in immunomodulatory functions. boosting a T cell-mediated immune response, Exemplary assays for transcription through and suppressing a T cell-mediated immune the NFAT response element that may be response. Additional highly preferred used or routinely modified to test NFAT- indications include inflammation and response element activity of polypeptides of inflammatory disorders. An additional highly the invention (including antibodies and preferred indication is infection (e.g., an agonists or antagonists of the invention) infectious disease as described below under include assays disclosed in Berger et al., “Infectious Disease”). Preferred indications Gene 66: 1-10 (1998); Cullen and Malm, include neoplastic diseases (e.g., leukemia, Methods in Enzymol 216: 362-368 (1992); lymphoma, and/or as described below under Henthorn et al., Proc Natl Acad Sci USA “Hyperproliferative Disorders”). Preferred 85: 6342-6346 (1988); Serfling et al., indications include neoplasms and cancers, such Biochim Biophys Acta 1498(1): 1-18 as, for example, leukemia, lymphoma, and (2000); De Boer et al., Int J Biochem Cell prostate, breast, lung, colon, pancreatic, Biol 31(10): 1221-1236 (1999); Fraser et al., esophageal, stomach, brain, liver and urinary Eur J Immunol 29(3): 838-844 (1999); and cancer. Other preferred indications include Yeseen et al., J Biol Chem 268(19): 14285-14293 benign dysproliferative disorders and pre- (1993), the contents of each of which neoplastic conditions, such as, for example, are herein incorporated by reference in its hyperplasia, metaplasia, and/or dysplasia. entirety. T cells that may be used according Preferred indications also include anemia, to these assays are publicly available (e.g., pancytopenia, leukopenia, thrombocytopenia, through the ATCC ™). Exemplary human T Hodgkin's disease, acute lymphocytic anemia cells that may be used according to these (ALL), plasmacytomas, multiple myeloma, assays include the JURKAT cell line, which Burkitt's lymphoma, arthritis, AIDS, is a suspension culture of leukemia cells granulomatous disease, inflammatory bowel that produce IL-2 when stimulated. disease, sepsis, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, asthma and allergy. 28 Activation of Assays for the activation of transcription Preferred embodiments of the invention include transcription through through the NFKB response element are using polypeptides of the invention (or NFKB response well-known in the art and may be used or antibodies, agonists, or antagonists thereof) in element in epithelial routinely modified to assess the ability of detection, diagnosis, prevention, and/or cells (such as HELA polypeptides of the invention (including treatment of Cancer, Wound Healing, and cells). antibodies and agonists or antagonists of the Inflamation. Highly preferred indications invention) to regulate NFKB transcription include neoplastic diseases (e.g., as described factors and modulate expression of below under “Hyperproliferative Disorders”). epithhelial genes. Exemplary assays for Highly preferred indications include neoplasms transcription through the NFKB response and cancers, such as, for example, melanoma, element that may be used or routinely and prostate, breast, lung, colon, pancreatic, modified to test NFKB-response element esophageal, stomach, brain, liver and urinary activity of polypeptides of the invention cancer. Other preferred indications include (including antibodies and agonists or benign dysproliferative disorders and pre- antagonists of the invention) include assays neoplastic conditions, such as, for example, disclosed in: Kaltschmidt B, et al., hyperplasia, metaplasia, and/or dysplasia. Oncogene, 18(21): 3213-3225 (1999); Beetz A, Preferred indications include include et al., Int J Radiat Biol, 76(11): 1443-1453 inflammation and inflammatory disorders. (2000); Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Valle Blazquez et al, Immunology 90(3): 455-460 (1997); Aramburau et al., J Exp Med 82(3): 801-810 (1995); and Fraser et al., 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. Epithelial cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary epithelial cells that may be used according to these assays include the HELA cell line. 29 Activation of Assays for the activation of transcription transcription through through the NFKB response element are NFKB response well-known in the art and may be used or element in immune routinely modified to assess the ability of cells (such as B- polypeptides of the invention (including cells). antibodies and agonists or antagonists of the invention) to regulate NFKB transcription factors and modulate expression of immunomodulatory genes. Exemplary assays for transcription through the NFKB response element that may be used or routinely modified to test NFKB-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Gri G, et al., Biol Chem, 273(11): 6431-6438 (1998); Pyatt DW, et al., Cell Biol Toxicol 2000; 16(1): 41-51 (2000); Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Valle Blazquez et al, Immunology 90(3): 455-460 (1997); Aramburau et al., J Exp Med 82(3): 801-810 (1995); and Fraser et al., 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. Immune cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary immune cells that may be used according to these assays include the Reh B-cell line. 30 Activation of Assays for the activation of transcription Preferred embodiments of the invention include transcription through through the NFKB response element are using polypeptides of the invention (or NFKB response well-known in the art and may be used or antibodies, agonists, or antagonists thereof) in element in immune routinely modified to assess the ability of detection, diagnosis, prevention, and/or cells (such as B- polypeptides of the invention (including treatment of Cancer, Autoimmunity, Allergy cells). antibodies and agonists or antagonists of the and Asthma invention) to regulate NFKB transcription factors and modulate expression of immunomodulatory genes. Exemplary assays for transcription through the NFKB response element that may be used or rountinely modified to test NFKB-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Gri G, et al., Biol Chem, 273(11): 6431-6438 (1998); Pyatt DW, et al., Cell Biol Toxicol 2000; 16(1): 41-51 (2000); Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Valle Blazquez et al, Immunology 90(3): 455-460 (1997); Aramburau et al., J Exp Med 82(3): 801-810 (1995); and Fraser et al., 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. Immune cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary immune cells that may be used according to these assays include the Reh B-cell line. 31 Activation of This reporter assay measures activation of Highly preferred indication includes allergy, transcription through the NFkB signaling pathway in Ku812 asthma, and rhinitis. Additional highly NFKB response human basophil cell line. Assays for the preferred indications include infection (e.g., an element in immune activation of transcription through the infectious disease as described below under cells (such as NFKB response element are well-known in “Infectious Disease”), and inflammation and basophils). the art and may be used or routinely inflammatory disorders. Preferred indications modified to assess the ability of include immunological and hempatopoietic polypeptides of the invention (including disorders (e.g., as described below under antibodies and agonists or antagonists of the “Immune Activity”, and “Blood-Related invention) to regulate NFKB transcription Disorders”). Preferred indications also include factors and modulate expression of autoimmune diseases (e.g., rheumatoid arthritis, immunomodulatory genes. Exemplary systemic lupus erythematosis, multiple sclerosis assays for transcription through the NFKB and/or as described below) and response element that may be used or immunodeficiencies (e.g., as described below). rountinely modified to test NFKB-response Preferred indications also include neoplastic element activity of polypeptides of the diseases (e.g., leukemia, lymphoma, melanoma, invention (including antibodies and agonists and/or as described below under or antagonists of the invention) include “Hyperproliferative Disorders”). Preferred assays disclosed in Berger et al., Gene 66: 1-10 indications include neoplasms and cancer, such (1998); Cullen and Malm, Methods in as, for example, leukemia, lymphoma, Enzymol 216: 362-368 (1992); Henthorn et melanoma, and prostate, breast, lung, colon, al., Proc Natl Acad Sci USA 85: 6342-6346 pancreatic, esophageal, stomach, brain, liver, (1988); Marone et al, Int Arch Allergy urinary tract cancers and as described below Immunol 114(3): 207-17 (1997), the under “Hyperproliferative Disorders”. contents of each of which are herein incorporated by reference in its entirety. Basophils that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary human basophil cell lines that may be used according to these assays include Ku812, originally established from a patient with chronic myelogenous leukemia. It is an immature prebasophilic cell line that can be induced to differentiate into mature basophils. 32 Activation of Assays for the activation of transcription Highly preferred indications include asthma, transcription through through the NFKB response element are allergy, hypersensitivity reactions, and NFKB response well-known in the art and may be used or inflammation. Preferred indications include element in immune routinely modified to assess the ability of infection (e.g., an infectious disease as cells (such as EOL1 polypeptides of the invention (including described below under “Infectious Disease”), cells). antibodies and agonists or antagonists of the immunological disorders, inflammation and invention) to regulate NFKB transcription inflammatory disorders (e.g., as described factors and modulate expression of below under “Immune Activity”, and “Blood- immunomodulatory genes. Exemplary Related Disorders”). Preferred indications assays for transcription through the NFKB include autoimmune diseases (e.g., rheumatoid response element that may be used or arthritis, systemic lupus erythematosis, multiple rountinely modified to test NFKB-response sclerosis and/or as described below) and element activity of polypeptides of the immunodeficiencies (e.g., as described below). invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Valle Blazquez et al, Immunology 90(3): 455-460 (1997); Aramburau et al., J Exp Med 82(3): 801-810 (1995); and Fraser et al., 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. For example, a reporter assay (which measures increases in transcription inducible from a NFkB responsive element in EOL-1 cells) may link the NFKB element to a repeorter gene and binds to the NFKB transcription factor, which is upregulated by cytokines and other factors. Exemplary immune cells that may be used according to these assays include eosinophils such as the human EOL-1 cell line of eosinophils. Eosinophils are a type of immune cell important in the allergic responses; they are recruited to tissues and mediate the inflammtory response of late stage allergic reaction. Eol-1 is a human eosinophil cell line. 33 Activation of This reporter assay measures activation of Highly preferred indication includes allergy, transcription through the NFkB signaling pathway in HMC-1 asthma, and rhinitis. Additional highly NFKB response human mast cell line. Activation of NFkB in preferred indications include infection (e.g., an element in immune mast cells has been linked to production of infectious disease as described below under cells (such as mast certain cytokines, such as IL-6 and IL-9. “Infectious Disease”), and inflammation and cells). Assays for the activation of transcription inflammatory disorders. Preferred indications through the NFKB response element are include immunological and hempatopoietic well-known in the art and may be used or disorders (e.g., as described below under routinely modified to assess the ability of “Immune Activity”, and “Blood-Related polypeptides of the invention (including Disorders”). Preferred indications also include antibodies and agonists or antagonists of the autoimmune diseases (e.g., rheumatoid arthritis, invention) to regulate NFKB transcription systemic lupus erythematosis, multiple sclerosis factors and modulate expression of and/or as described below) and immunomodulatory genes. Exemplary immunodeficiencies (e.g., as described below). assays for transcription through the NFKB Preferred indications also include neoplastic response element that may be used or diseases (e.g., leukemia, lymphoma, melanoma, rountinely modified to test NFKB-response and/or as described below under element activity of polypeptides of the “Hyperproliferative Disorders”). Preferred invention (including antibodies and agonists indications include neoplasms and cancer, such or antagonists of the invention) include as, for example, leukemia, lymphoma, assays disclosed in Berger et al., Gene 66: 1-10 melanoma, and prostate, breast, lung, colon, (1998); Cullen and Malm, Methods in pancreatic, esophageal, stomach, brain, liver, Enzymol 216: 362-368 (1992); Henthorn et urinary tract cancers and as described below al., Proc Natl Acad Sci USA 85: 6342-6346 under “Hyperproliferative Disorders”. (1988); Stassen et al, J Immunol 166(7): 4391-8 (2001); and Marquardt and Walker, J Allergy Clin Immunol 105(3): 500-5 (2000), the contents of each of which are herein incorporated by reference in its entirety. Mast cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary human mast cells that may be used according to these assays include the HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 34 Activation of Assays for the activation of transcription Highly preferred indications include transcription through through the NFKB response element are inflammation and inflammatory disorders. NFKB response well-known in the art and may be used or Highly preferred indications include blood element in immune routinely modified to assess the ability of disorders (e.g., as described below under cells (such as natural polypeptides of the invention (including “Immune Activity”, “Blood-Related killer cells). antibodies and agonists or antagonists of the Disorders”, and/or “Cardiovascular Disorders”). invention) to regulate NFKB transcription Highly preferred indications include factors and modulate expression of autoimmune diseases (e.g., rheumatoid arthritis, immunomodulatory genes. Exemplary systemic lupus erythematosis, multiple sclerosis assays for transcription through the NFKB and/or as described below), and response element that may be used or immunodeficiencies (e.g., as described below). rountinely modified to test NFKB-response An additional highly preferred indication is element activity of polypeptides of the infection (e.g., AIDS, and/or an infectious invention (including antibodies and agonists disease as described below under “Infectious or antagonists of the invention) include Disease”). Highly preferred indications assays disclosed in Berger et al., Gene 66: 1-10 include neoplastic diseases (e.g., melanoma, (1998); Cullen and Malm, Methods in leukemia, lymphoma, and/or as described Enzymol 216: 362-368 (1992); Henthorn et below under “Hyperproliferative Disorders”). al., Proc Natl Acad Sci USA 85: 6342-6346 Highly preferred indications include neoplasms (1988); Valle Blazquez et al, Immunology and cancers, such as, for example, melanoma, 90(3): 455-460 (1997); Aramburau et al., J renal cell carcinoma, leukemia, lymphoma, and Exp Med 82(3): 801-810 (1995); and Fraser prostate, breast, lung, colon, pancreatic, et al., 29(3): 838-844 (1999), the contents of esophageal, stomach, brain, liver and urinary each of which are herein incorporated by cancer. Other preferred indications include reference in its entirety. NK cells that may benign dysproliferative disorders and pre- be used according to these assays are neoplastic conditions, such as, for example, publicly available (e.g., through the hyperplasia, metaplasia, and/or dysplasia. ATCC ™). Exemplary NK cells that may be Preferred indications also include anemia, used according to these assays include the pancytopenia, leukopenia, thrombocytopenia, NK-YT cell line, which is a human natural Hodgkin's disease, acute lymphocytic anemia killer cell line with cytolytic and cytotoxic (ALL), plasmacytomas, multiple myeloma, activity. Burkitt's lymphoma, arthritis, AIDS, granulomatous disease, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, suppression of immune reactions to transplanted organs, asthma and allergy. 35 Activation of Assays for the activation of transcription Highly preferred indications include transcription through through the NFKB response element are inflammation and inflammatory disorders. NFKB response well-known in the art and may be used or Highly preferred indications include blood element in immune routinely modified to assess the ability of disorders (e.g., as described below under cells (such as T- polypeptides of the invention (including “Immune Activity”, “Blood-Related cells). antibodies and agonists or antagonists of the Disorders”, and/or “Cardiovascular Disorders”). invention) to regulate NFKB transcription Highly preferred indications include factors and modulate expression of autoimmune diseases (e.g., rheumatoid arthritis, immunomodulatory genes. Exemplary systemic lupus erythematosis, multiple sclerosis assays for transcription through the NFKB and/or as described below), and response element that may be used or immunodeficiencies (e.g., as described below). rountinely modified to test NFKB-response An additional highly preferred indication is element activity of polypeptides of the infection (e.g., AIDS, and/or an infectious invention (including antibodies and agonists disease as described below under “Infectious or antagonists of the invention) include Disease”). Highly preferred indications assays disclosed in Berger et al., Gene 66: 1-10 include neoplastic diseases (e.g., melanoma, (1998); Cullen and Malm, Methods in leukemia, lymphoma, and/or as described Enzymol 216: 362-368 (1992); Henthorn et below under “Hyperproliferative Disorders”). al., Proc Natl Acad Sci USA 85: 6342-6346 Highly preferred indications include neoplasms (1988); Black et al., Virus Gnes 15(2): 105-117 and cancers, such as, for example, melanoma, (1997); and Fraser et al., 29(3): 838-844 renal cell carcinoma, leukemia, lymphoma, and (1999), the contents of each of which are prostate, breast, lung, colon, pancreatic, herein incorporated by reference in its esophageal, stomach, brain, liver and urinary entirety. T cells that may be used according cancer. Other preferred indications include to these assays are publicly available (e.g., benign dysproliferative disorders and pre- through the ATCC ™). Exemplary human T neoplastic conditions, such as, for example, cells that may be used according to these hyperplasia, metaplasia, and/or dysplasia. assays include the SUPT cell line, which is Preferred indications also include anemia, a suspension culture of IL-2 and IL-4 pancytopenia, leukopenia, thrombocytopenia, responsive T cells. Additional exemplary Hodgkin's disease, acute lymphocytic anemia human T cells, such as the MOLT4, that (ALL), plasmacytomas, multiple myeloma, may be used according to these assays are Burkitt's lymphoma, arthritis, AIDS, publicly available (e.g., through the granulomatous disease, inflammatory bowel ATCC ™). disease, sepsis, neutropenia, neutrophilia, psoriasis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, suppression of immune reactions to transplanted organs, asthma and allergy. 36 Activation of Assays for the activation of transcription Highly preferred indications include transcription through through the NFKB response element are inflammation and inflammatory disorders. NFKB response well-known in the art and may be used or Highly preferred indications include element in immune routinely modified to assess the ability of immunological and hematopoietic disorders cells (such as the polypeptides of the invention (including (e.g., as described below under “Immune Jurkat human T cell antibodies and agonists or antagonists of the Activity”, “Blood-Related Disorders”, and/or line). invention) to regulate NFKB transcription “Cardiovascular Disorders”). Highly preferred factors and modulate expression of indications include autoimmune diseases (e.g., immunomodulatory genes. Exemplary rheumatoid arthritis, systemic lupus assays for transcription through the NFKB erythematosis, multiple sclerosis and/or as response element that may be used or described below), and immunodeficiencies rountinely modified to test NFKB-response (e.g., as described below). An additional highly element activity of polypeptides of the preferred indication is infection (e.g., AIDS, invention (including antibodies and agonists and/or an infectious disease as described below or antagonists of the invention) include under “Infectious Disease”). Highly preferred assays disclosed in Berger et al., Gene 66: 1-10 indications include neoplastic diseases (e.g., (1998); Cullen and Malm, Methods in melanoma, leukemia, lymphoma, and/or as Enzymol 216: 362-368 (1992); Henthorn et described below under “Hyperproliferative al., Proc Natl Acad Sci USA 85: 6342-6346 Disorders”). Highly preferred indications (1988); Valle Blazquez et al, Immunology include neoplasms and cancers, such 90(3): 455-460 (1997); Aramburau et al., J as, melanoma, renal cell carcinoma, leukemia, Exp Med 82(3): 801-810 (1995); and Fraser lymphoma, and prostate, breast, lung, colon, et al., 29(3): 838-844 (1999), the contents of pancreatic, esophageal, stomach, brain, liver each of which are herein incorporated by and urinary cancer. Other preferred indications reference in its entirety. T cells that may be include benign dysproliferative disorders and used according to these assays are publicly pre-neoplastic conditions, such as, for example, available (e.g., through the ATCC ™). T hyperplasia, metaplasia, and/or dysplasia. cells that may be used according to these Preferred indications also include anemia, assays are publicly available (e.g., through pancytopenia, leukopenia, thrombocytopenia, the ATCC ™). Exemplary human T cells Hodgkin's disease, acute lymphocytic anemia that may be used according to these assays (ALL), plasmacytomas, multiple myeloma, include the JURKAT cell line, which is a Burkitt's lymphoma, arthritis, AIDS, suspension culture of leukemia cells that granulomatous disease, inflammatory bowel produce IL-2 when stimulated. disease, sepsis, neutropenia, neutrophilia, psoriasis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, suppression of immune reactions to transplanted organs, asthma and allergy. 37 Activation of This assay uses a NFKB response element Highly preferred indications include transcription through (which will bind NFKB transcription inflammation and inflammatory disorders. NFKB response factors) linked to a reporter gene to measure Highly preferred indications include element in immune NFKB mediated transcription in the human immunological and hematopoietic disorders cells (such as the monocyte cell line U937. NFKB is (e.g., as described below under “Immune U937 human upregulated by cytokines and other factors Activity”, “Blood-Related Disorders”, and/or monocyte cell line). and NFKB element activation leads to “Cardiovascular Disorders”). Highly preferred expression of immunomodulatory genes. indications include autoimmune diseases (e.g., Activation of NFKB in monocytes can play rheumatoid arthritis, systemic lupus a role in immune responses. Exemplary erythematosis, multiple sclerosis and/or as assays for transcription through the NFKB described below), and immunodeficiencies response element that may be used or (e.g., as described below). An additional highly rountinely modified to test NFKB-response preferred indication is infection (e.g., AIDS, element activity of polypeptides of the and/or an infectious disease as described below invention (including antibodies and agonists under “Infectious Disease”). Highly preferred or antagonists of the invention) include indications include neoplastic diseases (e.g., assays disclosed in Berger et al., Gene 66: 1-10 melanoma, leukemia, lymphoma, and/or as (1998); Cullen and Malm, Methods in described below under “Hyperproliferative Enzymol 216: 362-368 (1992); Henthorn et Disorders”). Highly preferred indications al., Proc Natl Acad Sci USA 85: 6342-6346 include neoplasms and cancers, such (1988); Valle Blazquez et al, Immunology as, melanoma, renal cell carcinoma, leukemia, 90(3): 455-460 (1997); Aramburau et al., J lymphoma, and prostate, breast, lung, colon, Exp Med 82(3): 801-810 (1995); and Fraser pancreatic, esophageal, stomach, brain, liver et al., 29(3): 838-844 (1999), the contents of and urinary cancer. Other preferred indications each of which are herein incorporated by include benign dysproliferative disorders and reference in its entirety. Monocytic cells pre-neoplastic conditions, such as, for example, that may be used according to these assays hyperplasia, metaplasia, and/or dysplasia. are publicly available (e.g., through the Preferred indications also include anemia, ATCC ™). Exemplary human monocyte pancytopenia, leukopenia, thrombocytopenia, cells that may be used according to these Hodgkin's disease, acute lymphocytic anemia assays include the U937 cell line, which is (ALL), plasmacytomas, multiple myeloma, cell line derived by Sundstrom and Nilsson Burkitt's lymphoma, arthritis, AIDS, in 1974 from malignant cells obtained from granulomatous disease, inflammatory bowel the pleural effusion of a patient with disease, sepsis, neutropenia, neutrophilia, histiocytic lymphoma. psoriasis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, suppression of immune reactions to transplanted organs, asthma and allergy. 38 Activation of Assays for the activation of transcription transcription through through the NFKB response element are NFKB response well-known in the art and may be used or element in neuronal routinely modified to assess the ability of cells (such as polypeptides of the invention (including SKNMC cells). antibodies and agonists or antagonists of the invention) to regulate NFKB transcription factors and modulate expression of neuronal genes. Exemplary assays for transcription through the NFKB response element that may be used or routinely modified to test NFKB-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Gill JS, et al., Neurobiol Dis, 7(4): 448-461 (2000); Tamatani M, et al., J Biol Chem, 274(13): 8531-8538 (1999); Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Valle Blazquez et al, Immunology 90(3): 455-460 (1997); Aramburau et al., J Exp Med 82(3): 801-810 (1995); and Fraser et al., 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. Neuronal cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary neuronal cells that may be used according to these assays include the SKNMC neuronal cell line. 39 Activation of Assays for the activation of transcription Preferred embodiments of the invention include transcription through through the NFKB response element are using polypeptides of the invention (or NFKB response well-known in the art and may be used or antibodies, agonists, or antagonists thereof) in element in neuronal routinely modified to assess the ability of detection, diagnosis, prevention, and/or cells (such as polypeptides of the invention (including treatment of Neurological Diseases and SKNMC cells). antibodies and agonists or antagonists of the Disorders (e.g. Alzheimer″s Disease, invention) to regulate NFKB transcription Parkinson″s Disease, Brain Cancer, Seizures). factors and modulate expression of neuronal genes. Exemplary assays for transcription through the NFKB response element that may be used or routinely modified to test NFKB-response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Gill JS, et al., Neurobiol Dis, 7(4): 448-461 (2000); Tamatani M, et al., J Biol Chem, 274(13): 8531-8538 (1999); Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Valle Blazquez et al, Immunology 90(3): 455-460 (1997); Aramburau et al., J Exp Med 82(3): 801-810 (1995); and Fraser et al., 29(3): 838-844 (1999), the contents of each of which are herein incorporated by reference in its entirety. Neuronal cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary neuronal cells that may be used according to these assays include the SKNMC neuronal cell line. 40 Activation of Assays for the activation of transcription A preferred embodiment of the invention transcription through through the Serum Response Element includes a method for inhibiting (e.g., reducing) serum response (SRE) are well-known in the art and may be TNF alpha production. An alternative highly element in immune used or routinely modified to assess the preferred embodiment of the invention includes cells (such as natural ability of polypeptides of the invention a method for stimulating (e.g., increasing) TNF killer cells). (including antibodies and agonists or alpha production. Preferred indications antagonists of the invention) to regulate include blood disorders (e.g., as described serum response factors and modulate the below under “Immune Activity”, “Blood- expression of genes involved in growth and Related Disorders”, and/or “Cardiovascular upregulate the function of growth-related Disorders”), Highly preferred indications genes in many cell types. Exemplary assays include autoimmune diseases (e.g., rheumatoid for transcription through the SRE that may arthritis, systemic lupus erythematosis, Crohn″s be used or routinely modified to test SRE disease, multiple sclerosis and/or as described activity of the polypeptides of the invention below), immunodeficiencies (e.g., as described (including antibodies and agonists or below), boosting a T cell-mediated immune antagonists of the invention) include assays response, and suppressing a T cell-mediated disclosed in Berger et al., Gene 66: 1-10 immune response. Additional highly preferred (1998); Cullen and Malm, Methods in indications include inflammation and Enzymol 216: 362-368 (1992); Henthorn et inflammatory disorders, and treating joint al., Proc Natl Acad Sci USA 85: 6342-6346 damage in patients with rheumatoid arthritis. (1988); Benson et al., J Immunol An additional highly preferred indication is 153(9): 3862-3873 (1994); and Black et al., sepsis. Highly preferred indications include Virus Genes 12(2): 105-117 (1997), the neoplastic diseases (e.g., leukemia, lymphoma, content of each of which are herein and/or as described below under incorporated by reference in its entirety. T “Hyperproliferative Disorders”). Additionally, cells that may be used according to these highly preferred indications include neoplasms assays are publicly available (e.g., through and cancers, such as, for example, leukemia, the ATCC ™). Exemplary T cells that may lymphoma, melanoma, glioma (e.g., malignant be used according to these assays include glioma), solid tumors, and prostate, breast, the NK-YT cell line, which is a human lung, colon, pancreatic, esophageal, stomach, natural killer cell line with cytolytic and brain, liver and urinary cancer. Other preferred cytotoxic activity. indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Preferred indications include anemia, pancytopenia, leukopenia, thrombocytopenia, Hodgkin's disease, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, AIDS, granulomatous disease, inflammatory bowel disease, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, cardiac reperfusion injury, and asthma and allergy. An additional preferred indication is infection (e.g., an infectious disease as described below under “Infectious Disease”). 41 Activation of Assays for the activation of transcription A preferred embodiment of the invention transcription through through the Serum Response Element includes a method for inhibiting (e.g., reducing) serum response (SRE) are well-known in the art and may be TNF alpha production. An alternative preferred element in immune used or routinely modified to assess the embodiment of the invention includes a method cells (such as T- ability of polypeptides of the invention for stimulating (e.g., increasing) TNF alpha cells). (including antibodies and agonists or production. Preferred indications include antagonists of the invention) to regulate the blood disorders (e.g., as described below under serum response factors and modulate the “Immune Activity”, “Blood-Related expression of genes involved in growth. Disorders”, and/or ““Cardiovascular Exemplary assays for transcription through Disorders””), Highly preferred indications the SRE that may be used or routinely include autoimmune diseases (e.g., rheumatoid modified to test SRE activity of the arthritis, systemic lupus erythematosis, Crohn's polypeptides of the invention (including disease, multiple sclerosis and/or as described antibodies and agonists or antagonists of the below), immunodeficiencies (e.g., as described invention) include assays disclosed in below), boosting a T cell-mediated immune Berger et al., Gene 66: 1-10 (1998); Cullen response, and suppressing a T cell-mediated and Malm, Methods in Enzymol 216: 362-368 immune response. Additional highly preferred (1992); Henthorn et al., Proc Natl Acad indications include inflammation and Sci USA 85: 6342-6346 (1988); and Black et inflammatory disorders, and treating joint al., Virus Genes 12(2): 105-117 (1997), the damage in patients with rheumatoid arthritis. content of each of which are herein An additional highly preferred indication is incorporated by reference in its entirety. T sepsis. Highly preferred indications include cells that may be used according to these neoplastic diseases (e.g., leukemia, lymphoma, assays are publicly available (e.g., through and/or as described below under the ATCC ™). Exemplary mouse T cells “Hyperproliferative Disorders”). Additionally, that may be used according to these assays highly preferred indications include neoplasms include the CTLL cell line, which is an IL-2 and cancers, such as, for example, leukemia, dependent suspension culture of T cells with lymphoma, melanoma, glioma (e.g., malignant cytotoxic activity. Exemplary human T glioma), solid tumors, and prostate, breast, cells, such as the MOLT4, that may be used lung, colon, pancreatic, esophageal, stomach, according to these assays are publicly brain, liver and urinary cancer. Other preferred available (e.g., through the ATCC ™). indications include benign dysproliferative Additional exemplary human T cells that disorders and pre-neoplastic conditions, such may be used according to these assays as, for example, hyperplasia, metaplasia, and/or include the JURKAT cell line, which is a dysplasia. Preferred indications include suspension culture of leukemia cells that anemia, pancytopenia, leukopenia, produce IL-2 when stimulated. thrombocytopenia, Hodgkin's disease, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, AIDS, granulomatous disease, inflammatory bowel disease, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, cardiac reperfusion injury, and asthma and allergy. An additional preferred indication is infection (e.g., an infectious disease as described below under “Infectious Disease”). 42 Activation of Assays for the activation of transcription A highly preferred indication is obesity and/or transcription through through the Serum Response Element complications associated with obesity. serum response (SRE) are well-known in the art and may be Additional highly preferred indications include element in pre- used or routinely modified to assess the weight loss or alternatively, weight gain. An adipocytes. ability of polypeptides of the invention additional highly preferred indication is (including antibodies and agonists or diabetes mellitus. An additional highly antagonists of the invention) to regulate the preferred indication is a complication serum response factors and modulate the associated with diabetes (e.g., diabetic expression of genes involved in growth. retinopathy, diabetic nephropathy, kidney Exemplary assays for transcription through disease (e.g., renal failure, nephropathy and/or the SRE that may be used or routinely other diseases and disorders as described in the modified to test SRE activity of the “Renal Disorders” section below), diabetic polypeptides of the invention (including neuropathy, nerve disease and nerve damage antibodies and agonists or antagonists of the (e.g., due to diabetic neuropathy), blood vessel invention) include assays disclosed in blockage, heart disease, stroke, impotence (e.g., Berger et al., Gene 66: 1-10 (1998); Cullen due to diabetic neuropathy or blood vessel and Malm, Methods in Enzymol 216: 362-368 blockage), seizures, mental confusion, (1992); Henthorn et al., Proc Natl Acad drowsiness, nonketotic hyperglycemic- Sci USA 85: 6342-6346 (1988); and Black et hyperosmolar coma, cardiovascular disease al., Virus Genes 12(2): 105-117 (1997), the (e.g., heart disease, atherosclerosis, content of each of which are herein microvascular disease, hypertension, stroke, incorporated by reference in its entirety. and other diseases and disorders as described in Pre-adipocytes that may be used according the “Cardiovascular Disorders” section below), to these assays are publicly available (e.g., dyslipidemia, endocrine disorders (as described through the ATCC ™) and/or may be in the “Endocrine Disorders” section below), routinely generated. Exemplary mouse neuropathy, vision impairment (e.g., diabetic adipocyte cells that may be used according retinopathy and blindness), ulcers and impaired to these assays include 3T3-L1 cells. 3T3- wound healing, and infection (e.g., infectious L1 is an adherent mouse preadipocyte cell diseases and disorders as described in the line that is a continuous substrain of 3T3 “Infectious Diseases” section below). fibroblast cells developed through clonal Additional highly preferred indications are isolation and undergo a pre-adipocyte to complications associated with insulin adipose-like conversion under appropriate resistance. differentiation conditions known in the art. 43 Activation of Assays for the activation of transcription Highly preferred indications include allergy, transcription through through the Signal Transducers and asthma, and rhinitis. Additional highly STAT6 response Activators of Transcription (STAT6) preferred indications include infection (e.g., an element in immune response element in immune cells (such as infectious disease as described below under cells (such as mast in the human HMC-1 mast cell line) are “Infectious Disease”), and inflammation and cells). well-known in the art and may be used or inflammatory disorders. Preferred indications routinely modified to assess the ability of also include hematopoietic and immunological polypeptides of the invention (including disorders (e.g., as described below under antibodies and agonists or antagonists of the “Immune Activity”, “Blood-Related invention) to regulate STAT6 transcription Disorders”, and/or “Cardiovascular Disorders”), factors and modulate the expression of autoimmune diseases (e.g., rheumatoid arthritis, multiple genes. Exemplary assays for systemic lupus erythematosis, multiple sclerosis transcription through the STAT6 response and/or as described below), and element that may be used or routinely immunodeficiencies (e.g., as described below). modified to test STAT6 response element Preferred indications include neoplastic activity of the polypeptides of the invention diseases (e.g., leukemia, lymphoma, melanoma, (including antibodies and agonists or and/or as described below under antagonists of the invention) include assays “Hyperproliferative Disorders”). Preferred disclosed in Berger et al., Gene 66: 1-10 indications include neoplasms and cancer, such (1998); Cullen and Malm, Methods in as, for example, leukemia, lymphoma, Enzymol 216: 362-368 (1992); Henthorn et melanoma, and prostate, breast, lung, colon, al., Proc Natl Acad Sci USA 85: 6342-6346 pancreatic, esophageal, stomach, brain, liver (1988); Sherman, Immunol Rev 179: 48-56 and urinary cancer. Other preferred indications (2001); Malaviya and Uckun, J Immunol include benign dysproliferative disorders and 168: 421-426 (2002); Masuda et al., J Biol pre-neoplastic conditions, such as, for example, Chem 275(38): 29331-29337 (2000); and hyperplasia, metaplasia, and/or dysplasia. Masuda et al., J Biol Chem 276: 26107-26113 Preferred indications include hematopoietic and (2001), the contents of each of which immunological disorders such as arthritis, are herein incorporated by reference in its AIDS, granulomatous disease, inflammatory entirety. Mast cells that may be used bowel disease, sepsis, neutropenia, neutrophilia, according to these assays are publicly psoriasis, suppression of immune reactions to available (e.g., through the ATCC ™). transplanted organs and tissues, hemophilia, Exemplary human mast cells that may be hypercoagulation, diabetes mellitus, used according to these assays include the endocarditis, meningitis, and Lyme Disease. HMC-1 cell line, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 44 Activation of Assays for the activation of transcription transcription through through the Signal Transducers and STAT6 response Activators of Transcription (STAT6) element in immune response element are well-known in the art cells (such as natural and may be used or routinely modified to killer cells). assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate STAT6 transcription factors and modulate the expression of multiple genes. Exemplary assays for transcription through the STAT6 response element that may be used or routinely modified to test STAT6 response element activity of the polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Georas et al., Blood 92(12): 4529-4538 (1998); Moffatt et al., Transplantation 69(7): 1521-1523 (2000); Curiel et al., Eur J Immunol 27(8): 1982-1987 (1997); and Masuda et al., J Biol Chem 275(38): 29331-29337 (2000), the contents of each of which are herein incorporated by reference in its entirety. T cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary rat natural killer cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). 45 Activation of Assays for the activation of transcription A highly preferred indication is allergy. transcription through through the Signal Transducers and Another highly preferred indication is asthma. STAT6 response Activators of Transcription (STAT6) Additional highly preferred indications include element in immune response element are well-known in the art inflammation and inflammatory disorders. cells (such as T- and may be used or routinely modified to Preferred indications include blood disorders cells). assess the ability of polypeptides of the (e.g., as described below under “Immune invention (including antibodies and agonists Activity”, “Blood-Related Disorders”, and/or or antagonists of the invention) to regulate “Cardiovascular Disorders”). Preferred STAT6 transcription factors and modulate indications include autoimmune diseases (e.g., the expression of multiple genes. rheumatoid arthritis, systemic lupus Exemplary assays for transcription through erythematosis, multiple sclerosis and/or as the STAT6 response element that may be described below) and immunodeficiencies (e.g., used or routinely modified to test STAT6 as described below). Preferred response element activity of the indications include neoplastic diseases (e.g., polypeptides of the invention (including leukemia, lymphoma, melanoma, and/or as antibodies and agonists or antagonists of the described below under “Hyperproliferative invention) include assays disclosed in Disorders”). Preferred indications include Berger et al., Gene 66: 1-10 (1998); Cullen neoplasms and cancers, such as, leukemia, and Malm, Methods in Enzymol 216: 362-368 lymphoma, melanoma, and prostate, breast, (1992); Henthorn et al., Proc Natl Acad lung, colon, pancreatic, esophageal, stomach, Sci USA 85: 6342-6346 (1988); Georas et brain, liver and urinary cancer. Other preferred al., Blood 92(12): 4529-4538 (1998); indications include benign dysproliferative Moffatt et al., Transplantation 69(7): 1521-1523 disorders and pre-neoplastic conditions, such (2000); Curiel et al., Eur J Immunol as, for example, hyperplasia, metaplasia, and/or 27(8): 1982-1987 (1997); and Masuda et al., dysplasia. Preferred indications J Biol Chem 275(38): 29331-29337 (2000), include anemia, pancytopenia, leukopenia, the contents of each of which are herein thrombocytopenia, Hodgkin's disease, acute incorporated by reference in its entirety. T lymphocytic anemia (ALL), plasmacytomas, cells that may be used according to these multiple myeloma, Burkitt's lymphoma, assays are publicly available (e.g., through arthritis, AIDS, granulomatous disease, the ATCC ™). Exemplary T cells that may inflammatory bowel disease, sepsis, be used according to these assays include neutropenia, neutrophilia, psoriasis, suppression the SUPT cell line, which is a suspension of immune reactions to transplanted organs and culture of IL-2 and IL-4 responsive T cells. tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, and Lyme Disease. An additional preferred indication is infection (e.g., an infectious disease as described below under “Infectious Disease”). 46 Activation of Assays for the activation of transcription transcription through through the EGR response element are well- the EGR (Early known in the art and may be used or Growth Response) routinely modified to assess the ability of element in immune polypeptides of the invention (including cells (such as B- antibodies and agonists or antagonists of the cells). invention) to regulate EGR transcription factors and modulate expression of immunomodulatory genes. Exemplary assays for transcription through the EGR response element that may be used or routinely modified to test EGR response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Richards JD, et al., J Immunol, 166(6): 3855-3864 (2001); Dinkel, A, et al., J Exp Med, 188(12): 2215-2224 (1998); and, Newton, JS, et al., Eur J Immunol 1996 Apr; 26(4): 811-816 (1996), the contents of each of which are herein incorporated by reference in its entirety. Immune cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary epithelial cells that may be used according to these assays include the Raji cell line. 47 Activation of Assays for the activation of transcription Preferred embodiments of the invention include transcription through through the EGR response element are well- using polypeptides of the invention (or the EGR (Early known in the art and may be used or antibodies, agonists, or antagonists thereof) in Growth Response) routinely modified to assess the ability of detection, diagnosis, prevention, and/or element in immune polypeptides of the invention (including treatment of Cancer, Autoimmunity, Allergy cells (such as B- antibodies and agonists or antagonists of the and Asthma. cells). invention) to regulate EGR transcription factors and modulate expression of immunomodulatory genes. Exemplary assays for transcription through the EGR response element that may be used or routinely modified to test EGR response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in: Richards JD, et al., J Immunol, 166(6): 3855-3864 (2001); Dinkel, A, et al., J Exp Med, 188(12): 2215-2224 (1998); and, Newton, JS, et al., Eur J Immunol 1996 Apr; 26(4): 811-816 (1996), the contents of each of which are herein incorporated by reference in its entirety. Immune cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary epithelial cells that may be used according to these assays include the Raji cell line. 48 Activation or This reporter assay measures activation or inhibition of inhibition of the NFkB signaling pathway in transcription through Ku812 human basophil cell line. Assays for NFKB response the activation or inhibition of transcription element in immune through the NFKB response element are cells (such as well-known in the art and may be used or basophils). routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate NFKB transcription factors and modulate expression of immunomodulatory genes. NFkB is important in the pathogenesis of asthma. Exemplary assays for transcription through the NFKB response element that may be used or rountinely modified to test NFKB- response element activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Berger et al., Gene 66: 1-10 (1998); Cullen and Malm, Methods in Enzymol 216: 362-368 (1992); Henthorn et al., Proc Natl Acad Sci USA 85: 6342-6346 (1988); Marone et al, Int Arch Allergy Immunol 114(3): 207-17 (1997), the contents of each of which are herein incorporated by reference in its entirety. Cells were pretreated with SID supernatants or controls for 15-18 hours, and then 10 ng/mL of TNF was added to stimulate the NFkB reporter. SEAP activity was measured after 48 hours. Basophils that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary human basophil cell lines that may be used according to these assays include Ku812, originally established from a patient with chronic myelogenous leukemia. It is an immature prebasophilic cell line that can be induced to differentiate into mature basophils. See, Kishi et al., Leuk Res. 9: 381-390 (1985); Blom et al., Eur J Immunol. 22: 2025-32 (1992), where the contents of each are herein incorporated by reference in its entirety. 49 ATF-2 in CTLL-2 final_assay_desc final_assay_embodiment 50 Bone marrow cell Assay for measuring regulation of proliferation proliferation of mouse bone marrow cells (fibronectin (in the presence or absence of exogenous enhanced) Stem Cell Factor (SCF)) on a fibronectin extracellular matrix. Mouse bone marrow cells are plated onto 96-well fibronectin fragment coated plates in 0.2 ml of serum- free medium. Secreted protein factors (test factors) are tested with appropriate negative controls in the presence and absence of SCF (5.0 ng/ml), where secreted test factor supernates represent 10% of the total assay volume. The cells are grown for 7 days. The number of proliferating cells within the wells is quantitated by measuring thymidine incorporation into cellular DNA. This and similar assays may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate proliferation of bone marrow cells. Interactions between adhesion receptors on progenitor cells and their extracellular matrix ligands are essential for the control of hematopoiesis in bone marrow stroma. These interactions may help retain CD34+ hematopoietic progenitor cells within the an appropriate bone marrow environment, and adhesive interactions can also provide important costimulatory signals. As the ability of stem cells to undergo self-renewal in vitro is dependent upon their interaction with the stromal cells and the extracellular matrix (ECM), this assay identify factors which integrate with the ECM environment and are important for stimulating stem cell self- renewal. 51 Calcium flux in Assays for measuring calcium flux are well- Preferred embodiments of the invention include chondrocytes known in the art and may be used or using polypeptides of the invention (or routinely modified to assess the ability of antibodies, agonists, or antagonists thereof) in polypeptides of the invention (including detection, diagnosis, prevention, and/or antibodies and agonists or antagonists of the treatment of Bone and Cartilage Diseases, invention) to mobilize calcium. Cells including but not limited to Arthritis, Cartilige normally have very low concentrations of repair, Bone Repair, Osteoporosis, and related cytosolic calcium compared to much higher tumors including chondrosarcomas, extracellular calcium. Extracellular factors chondroblastomas, and chondromas. can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux in chondrocytes include assays disclosed in: Asada S, et al., Inflamm Res, 50(1): 19-23 (2001); Schwartz Z, et al., J Bone Miner Res, 6(7): 709-718 (1991); Iannotti JP, et al., J Bone Joint Surg Am, 67(1): 113-120 (1985); Sullivan E., et al., Methods Mol Biol 1999; 114: 125-133 (1999), the contents of each of which is herein incorporated by reference in its entirety. Cells that may be used according to these assays are publicly available (e.g., through the ATCC ™) and/or may be routinely generated. Exemplary cells that may be used according to these assays include bovine chondrocytes. 52 Calcium flux in Assays for measuring calcium flux are well- Preferred embodiments of the invention include immune cells (such known in the art and may be used or using polypeptides of the invention (or as monocytes) routinely modified to assess the ability of antibodies, agonists, or antagonists thereof) in polypeptides of the invention (including detection, diagnosis, prevention, and/or antibodies and agonists or antagonists of the treatment of Infection, Inflammation, invention) to mobilize calcium. Cells Atherosclerosis, Hypersensitivity, and normally have very low concentrations of Leukemias cytosolic calcium compared to much higher extracellular calcium. Extracellular factors can cause an influx of calcium, leading to activation of calcium responsive signaling pathways and alterations in cell functions. Exemplary assays that may be used or routinely modified to measure calcium flux in immune cells (such as monocytes) include assays disclosed in: Chan, CC, et al., J Pharmacol Exp Ther, 269(3): 891-896 (1994); Andersson, K, et al., Cytokine, 12(12): 1784-1787 (2000); Scully, SP, et al., J Clin Invest, 74(2) 589-599 (1984); and, Sullivan, E, et al., Methods Mol Biol, 114: 125-133 (1999), the contents of each of which is herein incorporated by reference in its entirety. Cells that may be used according to these assays are publicly available (e.g., through the ATCC ™) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the THP-1 monocyte cell line. 53 Caspase (+camptothecin) in SW480 54 Caspase (+paclitaxel) in SW480 55 CD152 in Human T cells 56 CD69 in Human T cells 57 CXCR4 in HT1080 58 CXCR4 in SW480 59 Endothelial Cell Caspase Apoptosis. Assays for caspase A highly preferred embodiment of the invention Apoptosis apoptosis are well known in the art and may includes a method for stimulating endothelial be used or routinely modified to assess the cell growth. An alternative highly preferred ability of polypeptides of the invention embodiment of the invention includes a method (including antibodies and agonists or for inhibiting endothelial cell growth. A antagonists of the invention) to promote highly preferred embodiment of the invention caspase protease-mediated apoptosis. includes a method for stimulating endothelial Induction of apoptosis in endothelial cells cell proliferation. An alternative highly supporting the vasculature of tumors is preferred embodiment of the invention includes associated with tumor regression due to loss a method for inhibiting endothelial cell of tumor blood supply. Exemplary assays proliferation. A highly preferred for caspase apoptosis that may be used or embodiment of the invention includes a method routinely modified to test capase apoptosis for stimulating apoptosis of endothelial cells. activity of polypeptides of the invention An alternative highly preferred embodiment of (including antibodies and agonists or the invention includes a method for inhibiting antagonists of the invention) include the (e.g., decreasing) apoptosis of endothelial cells. assays disclosed in Lee et al., FEBS Lett A highly preferred embodiment of the invention 485(2-3): 122-126 (2000); Nor et al., J Vasc includes a method for stimulating angiogenisis. Res 37(3): 209-218 (2000); and Karsan and An alternative highly preferred embodiment of Harlan, J Atheroscler Thromb 3(2): 75-80 the invention includes a method for inhibiting (1996); the contents of each of which are angiogenesis. A highly preferred herein incorporated by reference in its embodiment of the invention includes a method entirety. Endothelial cells that may be used for reducing cardiac hypertrophy. An according to these assays are publicly alternative highly preferred embodiment of the available (e.g., through commercial invention includes a method for inducing sources). Exemplary endothelial cells that cardiac hypertrophy. Highly preferred may be used according to these assays indications include neoplastic diseases (e.g., as include bovine aortic endothelial cells described below under “Hyperproliferative (bAEC), which are an example of Disorders”), and disorders of the cardiovascular endothelial cells which line blood vessels system (e.g., heart disease, congestive heart and are involved in functions that include, failure, hypertension, aortic stenosis, but are not limited to, angiogenesis, cardiomyopathy, valvular regurgitation, left vascular permeability, vascular tone, and ventricular dysfunction, atherosclerosis and immune cell extravasation. atherosclerotic vascular disease, diabetic nephropathy, intracardiac shunt, cardiac hypertrophy, myocardial infarction, chronic hemodynamic overload, and/or as described below under “Cardiovascular Disorders”). Highly preferred indications include cardiovascular, endothelial and/or angiogenic disorders (e.g., systemic disorders that affect vessels such as diabetes mellitus, as well as diseases of the vessels themselves, such as of the arteries, capillaries, veins and/or lymphatics). Highly preferred are indications that stimulate angiogenesis and/or cardiovascularization. Highly preferred are indications that inhibit angiogenesis and/or cardiovascularization. Highly preferred indications include antiangiogenic activity to treat solid tumors, leukemias, and Kaposi″s sarcoma, and retinal disorders. Highly preferred indications include neoplasms and cancer, such as, Kaposi″s sarcoma, hemangioma (capillary and cavernous), glomus tumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma, angiosarcoma, haemangiopericytoma, lymphangioma, lymphangiosarcoma. Highly preferred indications also include cancers such as, prostate, breast, lung, colon, pancreatic, esophageal, stomach, brain, liver, and urinary cancer. Preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Highly preferred indications also include arterial disease, such as, atherosclerosis, hypertension, coronary artery disease, inflammatory vasculitides, Reynaud″s disease and Reynaud″s phenomenom, aneurysms, restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular disorders such as peripheral vascular disease, and cancer. Highly preferred indications also include trauma such as wounds, burns, and injured tissue (e.g., vascular injury such as, injury resulting from balloon angioplasty, and atheroschlerotic lesions), implant fixation, scarring, ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal diseases such as acute renal failure, and osteoporosis. Additional highly preferred indications include stroke, graft rejection, diabetic or other retinopathies, thrombotic and coagulative disorders, vascularitis, lymph angiogenesis, sexual disorders, age-related macular degeneration, and treatment/ prevention of endometriosis and related conditions. Additional highly preferred indications include fibromas, heart disease, cardiac arrest, heart valve disease, and vascular disease. Preferred indications include blood disorders (e.g., as described below under “Immune Activity”, “Blood-Related Disorders”, and/or “Cardiovascular Disorders”). Preferred indications include autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis and/or as described below) and immunodeficiencies (e.g., as described below). Additional preferred indications include inflammation and inflammatory disorders (such as acute and chronic inflammatory diseases, e.g., inflammatory bowel disease and Crohn's disease), and pain management. 60 Glucose Production in H4IIE 61 Hexosaminidase in RBL-2H3 62 HLA-DR in Human T cells 63 ICAM in Normal Human Bronchial Epitheliae 64 ICAM in OE19 65 IFNg in Human T- cell 293T 66 IFNg in Human T- cell 2B9 67 IgG in Human B cells 68 IgG in Human B cells SAC 69 IL-10 in Human T- cell 293T 70 IL-10 in Human T- cell 2B9 71 IL-13 in HMC 72 IL-13 in Human T cells 73 IL-2 in Human T cells 74 IL-2 in Human T-cell 293T 75 IL-4 in HMC 76 IL-6 in HUVEC 77 IL-8 in Normal Human Bronchial Epitheliae 78 IL-8 in SW480 79 Inhibition of Kinase assay: measures the phosphorylation adipocyte ERK of Elk-1, an indication of activation of signaling pathway. extracellular signal regulated kinase (ERK). ERK pathway regulates cell growth, proliferation and differentiation. Cells were pretreated with SID supernatants for 15-18 hours, and then 100 nM of insulin was added to stimulate ERK kinase. Phosphorylation of Elk-1 was measured after a 20 minute incubation. Pre- adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC ™) and/or may be routinely generated. Exemplary mouse adipocyte cells that may be used according to these assays include 3T3-L1 cells. 3T3- L1 is an adherent mouse preadipocyte cell line that is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation and undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation conditions known in the art. Cells were differentiated to an adipose-like state before being used in the screen. See Green et al., Cell 3: 127-133 (1974), the contents of which are herein incorporated by reference in its entirety. 80 Inhibition of squalene Reporter Assay: construct contains synthetase gene regulatory and coding sequence of squalene transcription. synthetase, the first specific enzyme in the cholesterol biosynthetic pathway. See Jiang, et al., J. Biol. Chem. 268: 12818-128241 (993), the contents of which are herein incorporated by reference in its entirety. Cells were treated with SID supernatants, and SEAP activity was measured after 72 hours. HepG2 is a human hepatocellular carcinoma cell line (ATCC ™ HB-8065). See Knowles et al., Science. 209: 497-9 (1980), the contents of which are herein incorporated by reference in its entirety. 81 Insulin Secretion Assays for measuring secretion of insulin A highly preferred indication is diabetes are well-known in the art and may be used mellitus. An additional highly preferred or routinely modified to assess the ability of indication is a complication associated with polypeptides of the invention (including diabetes (e.g., diabetic retinopathy, diabetic antibodies and agonists or antagonists of the nephropathy, kidney disease (e.g., renal failure, invention) to stimulate insulin secretion. nephropathy and/or other diseases and disorders For example, insulin secretion is measured as described in the “Renal Disorders” section by FMAT using anti-rat insulin antibodies. below), diabetic neuropathy, nerve disease and Insulin secretion from pancreatic beta cells nerve damage (e.g., due to diabetic neuropathy), is upregulated by glucose and also by blood vessel blockage, heart disease, stroke, certain proteins/peptides, and disregulation impotence (e.g., due to diabetic neuropathy or is a key component in diabetes. Exemplary blood vessel blockage), seizures, mental assays that may be used or routinely confusion, drowsiness, nonketotic modified to test for stimulation of insulin hyperglycemic-hyperosmolar coma, secretion (from pancreatic cells) by cardiovascular disease (e.g., heart disease, polypeptides of the invention (including atherosclerosis, microvascular disease, antibodies and agonists or antagonists of the hypertension, stroke, and other diseases and invention) include assays disclosed in: disorders as described in the “Cardiovascular Shimizu, H., et al., Endocr J, 47(3): 261-9 Disorders” section below), dyslipidemia, (2000); Salapatek, A. M., et al., Mol endocrine disorders (as described in the Endocrinol, 13(8): 1305-17 (1999); “Endocrine Disorders” section below), Filipsson, K., et al., Ann N Y Acad Sci, neuropathy, vision impairment (e.g., diabetic 865: 441-4 (1998); Olson, L. K., et al., J Biol retinopathy and blindness), ulcers and impaired Chem, 271(28): 16544-52 (1996); and, wound healing, and infection (e.g., infectious Miraglia S et. al., Journal of Biomolecular diseases and disorders as described in the Screening, 4: 193-204 (1999), the contents “Infectious Diseases” section below, especially of each of which is herein incorporated by of the urinary tract and skin), carpal tunnel reference in its entirety. Pancreatic cells syndrome and Dupuytren's contracture). that may be used according to these assays An additional highly preferred indication is are publicly available (e.g., through the obesity and/or complications associated with ATCC ™) and/or may be routinely obesity. Additional highly preferred indications generated. Exemplary pancreatic cells that include weight loss or alternatively, weight may be used according to these assays gain. Additional highly preferred indications are include HITT15 Cells. HITT15 are an complications associated with insulin adherent epithelial cell line established from resistance. Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 82 MCP-1 in Eol-1 83 MCP-1 in HUVEC 84 MIP-1a in HMC 85 Myoblast cell Assays for muscle cell proliferation are well Highly preferred indications include diabetes, proliferation known in the art and may be used or myopathy, muscle cell atrophy, cancers of routinely modified to assess the ability of muscle (such as, rhabdomyoma, and polypeptides of the invention (including rhabdosarcoma), cardiovascular disorders (such antibodies and agonists or antagonists of the as congestive heart failure, cachexia, myxomas, invention) to stimulate or inhibit myoblast fibromas, congenital cardiovascular cell proliferation. Exemplary assays for abnormalities, heart disease, cardiac arrest, myoblast cell proliferation that may be used heart valve disease, vascular disease, and also or routinely modified to test activity of as described below under “Cardiovascular polypeptides and antibodies of the invention Disorders”), stimulating myoblast proliferation, (including agonists or antagonists of the and inhibiting myoblast proliferation. invention) include, for example, assays disclosed in: Soeta, C., et al. “Possible role for the c-ski gene in the proliferation of myogenic cells in regenerating skeletal muscles of rats” Dev Growth Differ Apr; 43(2): 155-64 (2001); Ewton DZ, et al., “IGF binding proteins-4, -5 and -6 may play specialized roles during L6 myoblast proliferation and differentiation” J Endocrinol Mar; 144(3): 539-53 (1995); and, Pampusch MS, et al., “Effect of transforming growth factor beta on proliferation of L6 and embryonic porcine myogenic cells” J Cell Physiol Jun; 143(3): 524-8 (1990); the contents of each of which are herein incorporated by reference in their entirety. Exemplary myoblast cells that may be used according to these assays include the rat myoblast L6 cell line. Rat myoblast L6 cells are an adherent rat myoblast cell line, isolated from primary cultures of rat thigh muscle, that fuse to form multinucleated myotubes and striated fibers after culture in differentiation media. 86 Production of GM- GM-CSF FMAT. GM-CSF is expressed by A highly preferred embodiment of the invention CSF activated T cells, macrophages, endothelial includes a method for stimulating the cells, and fibroblasts. GM-CSF regulates production of GM-CSF. An alternative highly differentiation and proliferation of preferred embodiment of the invention includes granulocytes-macrophage progenitors and a method for inhibiting the production of GM- enhances antimicrobial activity in CSF. Highly preferred indications include neutrophils, monocytes and macrophage. inflammation and inflammatory disorders. An Additionally, GM-CSF plays an important additional highly preferred indication is role in the differentiation of dendritic cells infection (e.g., as described below under and monocytes, and increases antigen “Infectious Disease”. Highly preferred presentation. GM-CSF is considered to be a indications include blood disorders (e.g., proinflammatory cytokine. Assays for neutropenia (and the prevention of neutropenia immunomodulatory proteins that promote (e.g., in HIV infected patients), and/or as the production of GM-CSF are well known described below under “Immune Activity”, in the art and may be used or routinely “Blood-Related Disorders”, and/or modified to assess the ability of “Cardiovascular Disorders”). Highly preferred polypeptides of the invention (including indications also include autoimmune diseases antibodies and agonists or antagonists of the (e.g., rheumatoid arthritis, systemic lupus invention) to mediate immunomodulation erythematosis, multiple sclerosis and/or as and modulate the growth and differentiation described below) and immunodeficiencies (e.g., of leukocytes. Exemplary assays that test as described below). Additional highly for immunomodulatory proteins evaluate preferred indications include asthma. Highly the production of cytokines, such as GM- preferred indications include neoplastic diseases CSF, and the activation of T cells. Such (e.g., leukemia (e.g., acute lymphoblastic assays that may be used or routinely leukemia, and acute myelogenous leukemia), modified to test immunomodulatory activity lymphoma (e.g., non-Hodgkin″s lymphoma and of polypeptides of the invention (including Hodgkin″s disease), and/or as described below antibodies and agonists or antagonists of the under “Hyperproliferative Disorders”). Highly invention) include the assays disclosed in preferred indications include neoplasms and Miraglia et al., J Biomolecular Screening cancers, such as, leukemia, lymphoma, 4: 193-204 (1999); Rowland et al., melanoma, and prostate, breast, lung, colon, “Lymphocytes: a practical approach” pancreatic, esophageal, stomach, brain, liver Chapter 6: 138-160 (2000); and Ye et al., J and urinary cancer. Other preferred indications Leukoc Biol (58(2): 225-233, the contents of include benign dysproliferative disorders and each of which are herein incorporated by pre-neoplastic conditions, such as, for example, reference in its entirety. Natural killer cells hyperplasia, metaplasia, and/or dysplasia. that may be used according to these assays Highly preferred indications include: are publicly available (e.g., through the suppression of immune reactions to ATCC ™) or may be isolated using transplanted organs and tissues (e.g., bone techniques disclosed herein or otherwise marrow transplant); accelerating myeloid known in the art. Natural killer (NK) cells recovery; and mobilizing hematopoietic are large granular lymphocytes that have progenitor cells. Preferred indications include cytotoxic activity but do bind antigen. NK boosting a T cell-mediated immune response, cells show antibody-independent killing of and alternatively, suppressing a T cell-mediated tumor cells and also recognize antibody immune response. Preferred indications bound on target cells, via NK Fc receptors, include anemia, pancytopenia, leukopenia, leading to cell-mediated cytotoxicity. thrombocytopenia, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, AIDS, granulomatous disease, inflammatory bowel disease, sepsis, neutrophilia, psoriasis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and allergy. 87 Production of ICAM Endothelial cells, which are cells that line Highly preferred indications include in endothelial cells blood vessels, and are involved in functions inflammation (acute and chronic), restnosis, (such as human that include, but are not limited to, atherosclerosis, asthma and allergy. Highly umbilical vein angiogenesis, vascular permeability, preferred indications include inflammation and endothelial cells vascular tone, and immune cell inflammatory disorders, immunological (HUVEC)) extravasation. Exemplary endothelial cells disorders, neoplastic disorders (e.g. that may be used in ICAM production cancer/tumorigenesis), and cardiovascular assays include human umbilical vein disorders (such as described below under endothelial cells (HUVEC), and are “Immune Activity”, “Blood-Related available from commercial sources. The Disorders”, “Hyperproliferative Disorders” expression of ICAM (CD54), a intergral and/or “Cardiovascular Disorders”). Highly membrane protein, can be upregulated by preferred indications include neoplasms and cytokines or other factors, and ICAM cancers such as, for example, leukemia, expression is important in mediating lymphoma, melanoma, renal cell carcinoma, immune and endothelial cell interactions and prostate, breast, lung, colon, pancreatic, leading to immune and inflammatory esophageal, stomach, brain, liver and urinary responses. Assays for measuring expression cancer. Other preferred indications include of ICAM-1 are well-known in the art and benign dysproliferative disorders and pre- may be used or routinely modified to assess neoplastic conditions, such as, for example, the ability of polypeptides of the invention hyperplasia, metaplasia, and/or dysplasia. (including antibodies and agonists or antagonists of the invention) to regulate ICAM-1 expression. Exemplary assays that may be used or routinely modified to measure ICAM-1 expression include assays disclosed in: Rolfe BE, et al., Atherosclerosis, 149(1): 99-110 (2000); Panettieri RA Jr, et al., J Immunol, 154(5): 2358-2365 (1995); and, Grunstein MM, et al., Am J Physiol Lung Cell Mol Physiol, 278(6): L1154-L1163 (2000), the contents of each of which is herein incorporated by reference in its entirety. 88 Production of ICAM-1 Assays for measuring expression of ICAM- Preferred embodiments of the invention include 1 are well-known in the art and may be used using polypeptides of the invention (or or routinely modified to assess the ability of antibodies, agonists, or antagonists thereof) in polypeptides of the invention (including detection, diagnosis, prevention, and/or antibodies and agonists or antagonists of the treatment of Vascular Disease, Atherosclerosis, invention) to regulate ICAM-1 expression. Restenosis, Stroke, and Asthma. Exemplary assays that may be used or routinely modified to measure ICAM-1 expression include assays disclosed in: Rolfe BE, et al., Atherosclerosis, 149(1): 99-110 (2000); Panettieri RA Jr, et al., J Immunol, 154(5): 2358-2365 (1995); and, Grunstein MM, et al., Am J Physiol Lung Cell Mol Physiol, 278(6): L1154-L1163 (2000), the contents of each of which is herein incorporated by reference in its entirety. Cells that may be used according to these assays are publicly available (e.g., through the ATCC ™) and/or may be routinely generated. Exemplary cells that may be used according to these assays include Aortic Smooth Muscle Cells (AOSMC); such as bovine AOSMC. Exemplary cells that may be used according to these assays include microvascular endothelial cells (MVEC). 89 Production of IFNgamma FMAT. IFNg plays a central A highly preferred embodiment of the invention IFNgamma using a T role in the immune system and is considered includes a method for stimulating the cells to be a proinflammatory cytokine. IFNg production of IFNg. An alternative highly promotes TH1 and inhibits TH2 preferred embodiment of the invention includes differentiation; promotes IgG2a and inhibits a method for inhibiting the production of IFNg. IgE secretion; induces macrophage Highly preferred indications include blood activation; and increases MHC expression. disorders (e.g., as described below under Assays for immunomodulatory proteins “Immune Activity”, “Blood-Related produced by T cells and NK cells that Disorders”, and/or ““Cardiovascular regulate a variety of inflammatory activities Disorders””), and infection (e.g., viral and inhibit TH2 helper cell functions are infections, tuberculosis, infections associated well known in the art and may be used or with chronic granulomatosus disease and routinely modified to assess the ability of malignant osteoporosis, and/or as described polypeptides of the invention (including below under “Infectious Disease”). Highly antibodies and agonists or antagonists of the preferred indications include autoimmune invention) to mediate immunomodulation, disease (e.g., rheumatoid arthritis, systemic regulate inflammatory activities, modulate lupus erythematosis, multiple sclerosis and/or TH2 helper cell function, and/or mediate as described below), immunodeficiency (e.g., as humoral or cell-mediated immunity. described below), boosting a T cell-mediated Exemplary assays that test for immune response, and suppressing a T cell- immunomodulatory proteins evaluate the mediated immune response. Additional highly production of cytokines, such as Interferon preferred indications include inflammation and gamma (IFNg), and the activation of T inflammatory disorders. Additional preferred cells. Such assays that may be used or indications include idiopathic pulmonary routinely modified to test fibrosis. Highly preferred indications include immunomodulatory activity of polypeptides neoplastic diseases (e.g., leukemia, lymphoma, of the invention (including antibodies and melanoma, and/or as described below under agonists or antagonists of the invention) “Hyperproliferative Disorders”). Highly include the assays disclosed in Miraglia et preferred indications include neoplasms and al., J Biomolecular Screening 4: 193-204 cancers, such as, for example, leukemia, (1999); Rowland et al., ““Lymphocytes: a lymphoma, melanoma, and prostate, breast, practical approach”” Chapter 6: 138-160 lung, colon, pancreatic, esophageal, stomach, (2000); Gonzalez et al., J Clin Lab Anal brain, liver and urinary cancer. Other preferred 8(5): 225-233 (1995); Billiau et al., Ann NY indications include benign dysproliferative Acad Sci 856: 22-32 (1998); Boehm et al., disorders and pre-neoplastic conditions, such Annu Rev Immunol 15: 749-795 (1997), and as, for example, hyperplasia, metaplasia, and/or Rheumatology (Oxford) 38(3): 214-20 dysplasia. Preferred indications include (1999), the contents of each of which are anemia, pancytopenia, leukopenia, herein incorporated by reference in its thrombocytopenia, Hodgkin's disease, acute entirety. Human T cells that may be used lymphocytic anemia (ALL), plasmacytomas, according to these assays may be isolated multiple myeloma, Burkitt's lymphoma, using techniques disclosed herein or arthritis, AIDS, granulomatous disease, otherwise known in the art. Human T cells inflammatory bowel disease, sepsis, are primary human lymphocytes that mature neutropenia, neutrophilia, psoriasis, suppression in the thymus and express a T Cell receptor of immune reactions to transplanted organs and and CD3, CD4, or CD8. These cells tissues, hemophilia, hypercoagulation, diabetes mediate humoral or cell-mediated immunity mellitus, endocarditis, meningitis, Lyme and may be preactivated to enhance Disease, asthma and allergy. responsiveness to immunomodulatory factors. 90 Production of IFNgamma FMAT. IFNg plays a central IFNgamma using role in the immune system and is considered Natural Killer cells to be a proinflammatory cytokine. IFNg promotes TH1 and inhibits TH2; promotes IgG2a and inhibits IgE; induces macrophage activation; and increases MHC expression. Assays for immunomodulatory proteins produced by T cells and NK cells that regulate a variety of inflammatory activities and inhibit TH2 helper cell functions are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, regulate inflammatory activities, modulate TH2 helper cell function, and/or mediate humoral or cell- mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as Interferon gamma (IFNg), and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Gonzalez et al., J Clin Lab Anal 8(5): 225-233 (1995); Billiau et al., Ann NY Acad Sci 856: 22-32 (1998); Boehm et al., Annu Rev Immunol 15: 749-795 (1997), and Rheumatology (Oxford) 38(3): 214-20 (1999), the contents of each of which are herein incorporated by reference in its entirety. Natural Killer (NK) cells that may be used according to these assays are publicly available (e.g., through the ATCC ™) or may be isolated using techniques disclosed herein or otherwise known in the art. Natural killer (NK) cells are large granular lymphocytes that have cytotoxic activity but do bind antigen. NK cells show antibody-independent killing of tumor cells and also recognize antibody bound on target cells, via NK Fc receptors, leading to cell-mediated cytotoxicity. 91 Production of IL-10 Assays for production of IL-10 and Highly preferred indications include allergy and and activation of T- activation of T-cells are well known in the asthma. Additional highly preferred indications cells. art and may be used or routinely modified to include immune and hematopoietic disorders assess the ability of polypeptides of the (e.g., as described below under “Immune invention (including antibodies and agonists Activity”, and “Blood-Related Disorders”), or antagonists of the invention) to stimulate autoimmune diseases (e.g., rheumatoid arthritis, or inhibit production of IL-10 and/or systemic lupus erythematosis, Crohn″s disease, activation of T-cells. Exemplary assays that multiple sclerosis and/or as described below), may be used or routinely modified to assess immunodeficiencies (e.g., as described below), the ability of polypeptides and antibodies of boosting a T cell-mediated immune response, the invention (including agonists or and suppressing a T cell-mediated immune antagonists of the invention) to modulate response. IL-10 production and/or T-cell proliferation include, for example, assays such as disclosed and/or cited in: Robinson, DS, et al., “Th-2 cytokines in allergic disease” Br Med Bull; 56 (4): 956-968 (2000), and Cohn, et al., “T-helper type 2 cell-directed therapy for asthma” Pharmacology & Therapeutics; 88: 187-196 (2000); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL10 secreted from Th2 cells may be measured as a marker of Th2 cell activation. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated via in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 92 Production of IL-10 IL-10 FMAT. Assays for and downregulation immunomodulatory proteins produced by of immune responses activated T cells, B cells, and monocytes that exhibit anti-inflammatory activity and downregulate monocyte/macrophage function and expression of cytokines are well known in the art and may be used or routinely modified to assess the ability of the polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, regulate inflammatory activities, and modulate immune cell function and cytokine production. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as IL-10, and the downmodulation of immune responses. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); and Koning et al., Cytokine 9(6): 427-436 (1997), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 93 Production of IL-13 IL-13 FMAT. IL-13 enhances IgM, IgG, and IgE production and induces FcER1. IL- 13 has anti-inflammatory activity on monocytes and macrophages. Assays for immunomodulatory proteins produced by T cells that inhibit activation and release of cytokines by macrophages are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, regulate cytokine release, stimulate immune cells through the binding of IL-13 and IL-4 receptors, and/or mediate humoral or cell- mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as IL-13, the inhibition of cytokines released by macrophages. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); and Ohshima et al., Blood 92(9): 3338-3345 (1998), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell- mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 94 Production of IL-13 Assays for production of IL-13 and and activation of T- activation of T-cells are well known in the cells. art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to stimulate or inhibit production of IL-13 and/or activation of T-cells. Exemplary assays for IL-13 production that may be used or routinely modified to test activity of polypeptides and antibodies of the invention (including agonists or antagonists of the invention) include, for example, assays such as disclosed and/or cited in: Grunig, G, et al., “Requirement for IL-13 independently of IL-4 in Experimental asthma” Science; 282: 2261-2263 (1998), and Wills- Karp M, et al., “Interleukin-13: central mediator of allergic asthma” Science; 282: 2258-2261 (1998); the contents of each of which are herein incorporated by reference in their entirety. Exemplary cells that may be used according to these assays include Th2 cells. IL13, a Th2 type cytokine, is a potent stimulus for mucus production, airway hyper-responsiveness and allergic asthma. Th2 cells are a class of T cells that secrete IL4, IL10, IL13, IL5 and IL6. Factors that induce differentiation and activation of Th2 cells play a major role in the initiation and pathogenesis of allergy and asthma. Primary T helper 2 cells are generated in in vitro culture under Th2 polarizing conditions using peripheral blood lymphocytes isolated from cord blood. 95 Production of IL-2 IL-2 FMAT. IL-2 is the principal T cell and activation of T factor that allows T cell expansion and cells differentiation into effector cells. Assays for immunomodulatory proteins secreted by TH1 cells that promote T cell and NK cell growth and differentiation are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, promote immune cell growth and differentiation, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as IL-2, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Laduda et al., Immunology 94(4): 496-502 (1998); and Powell et al., Immunol Rev 165: 287-300 (1998), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 96 Production of IL-4 IL-4 FMAT. Assays for A highly preferred embodiment of the invention immunomodulatory proteins secreted by includes a method for stimulating (e.g., TH2 cells that stimulate B cells, T cells, increasing) IL-4 production. An alternative macrophages and mast cells and promote highly preferred embodiment of the invention polarization of CD4+ cells into TH2 cells includes a method for inhibiting (e.g., reducing) are well known in the art and may be used IL-4 production. A highly preferred or routinely modified to assess the ability of indication includes asthma. A highly polypeptides of the invention (including preferred indication includes allergy. A antibodies and agonists or antagonists of the highly preferred indication includes rhinitis. invention) to mediate immunomodulation, Additional highly preferred indications include stimulate immune cells, modulate immune inflammation and inflammatory disorders. cell polarization, and/or mediate humoral or Highly preferred indications include neoplastic cell-mediated immunity. Exemplary assays diseases (e.g., leukemia, lymphoma, that test for immunomodulatory proteins melanoma, and/or as described below under evaluate the production of cytokines, such “Hyperproliferative Disorders”). Preferred as IL-4, and the stimulation of immune indications include neoplasms and cancers, such cells, such as B cells, T cells, macrophages as, for example, leukemia, lymphoma, and mast cells. Such assays that may be melanoma, and prostate, breast, lung, colon, used or routinely modified to test pancreatic, esophageal, stomach, brain, liver immunomodulatory activity of polypeptides and urinary cancer. Other preferred indications of the invention (including antibodies and include benign dysproliferative disorders and agonists or antagonists of the invention) pre-neoplastic conditions, such as, for example, include the assays disclosed in Miraglia et hyperplasia, metaplasia, and/or dysplasia. al., J Biomolecular Screening 4: 193-204 Preferred indications include blood disorders (1999); Rowland et al., “Lymphocytes: a (e.g., as described below under “Immune practical approach” Chapter 6: 138-160 Activity”, “Blood-Related Disorders”, and/or (2000); Gonzalez et al., J Clin Lab Anal “Cardiovascular Disorders”). Preferred 8(5): 277-283 (1194); Yssel et al., Res indications include autoimmune diseases (e.g., Immunol 144(8): 610-616 (1993); Bagley et rheumatoid arthritis, systemic lupus al., Nat Immunol 1(3): 257-261 (2000); and erythematosis, multiple sclerosis and/or as van der Graaff et al., Rheumatology described below) and immunodeficiencies (e.g., (Oxford) 38(3): 214-220 (1999), the contents as described below). Preferred indications of each of which are herein incorporated by include anemia, pancytopenia, leukopenia, reference in its entirety. Human T cells that thrombocytopenia, Hodgkin's disease, acute may be used according to these assays may lymphocytic anemia (ALL), plasmacytomas, be isolated using techniques disclosed multiple myeloma, Burkitt's lymphoma, herein or otherwise known in the art. arthritis, AIDS, granulomatous disease, Human T cells are primary human inflammatory bowel disease, sepsis, lymphocytes that mature in the thymus and neutropenia, neutrophilia, psoriasis, suppression express a T cell receptor and CD3, CD4, or of immune reactions to transplanted organs and CD8. These cells mediate humoral or cell- tissues, hemophilia, hypercoagulation, diabetes mediated immunity and may be preactivated mellitus, endocarditis, meningitis, and Lyme to enhance responsiveness to Disease. An additonal preferred indication immunomodulatory factors. is infection (e.g., an infectious disease as described below under “Infectious Disease”). 97 Production of IL-5 IL-5 FMAT. Assays for A highly preferred embodiment of the invention immunomodulatory proteins secreted by includes a method for inhibiting (e.g., reducing) TH2 cells, mast cells, basophils, and IL-5 production. An alternative highly preferred eosinophils that stimulate eosinophil embodiment of the invention includes a method function and B cell Ig production and for stimulating (e.g., increasing) IL-5 promote polarization of CD4+ cells into production. A highly preferred embodiment TH2 cells are well known in the art and may of the invention includes a method for be used or routinely modified to assess the stimulating (e.g., increasing) immunoglobulin ability of polypeptides of the invention production. An alternative highly preferred (including antibodies and agonists or embodiment of the invention includes a method antagonists of the invention) to mediate for inhibiting (e.g., decreasing) immunomodulation, stimulate immune cell immunoglobulin production. A highly function, modulate B cell Ig production, preferred indication includes allergy. A modulate immune cell polarization, and/or highly preferred indication includes asthma. mediate humoral or cell-mediated A highly preferred indication includes rhinitis. immunity. Exemplary assays that test for An additional highly preferred indication is immunomodulatory proteins evaluate the infection (e.g., an infectious disease as production of cytokines, such as IL-5, and described below under ““Infectious Disease””), the stimulation of eosinophil function and B and inflammation and inflammatory disorders. cell Ig production. Such assays that may be Preferred indications include blood disorders used or routinely modified to test (e.g., as described below under “Immune immunomodulatory activity of polypeptides Activity”, “Blood-Related Disorders”, and/or of the invention (including antibodies and ““Cardiovascular Disorders””). Preferred agonists or antagonists of the invention) indications include autoimmune diseases (e.g., include the assays disclosed in Miraglia et rheumatoid arthritis, systemic lupus al., J Biomolecular Screening 4: 193-204 erythematosis, multiple sclerosis and/or as (1999); Rowland et al., ““Lymphocytes: a described below) and immunodeficiencies (e.g., practical approach”” Chapter 6: 138-160 as described below). Preferred indications (2000); Ohshima et al., Blood 92(9): 3338-3345 include neoplastic diseases (e.g., leukemia, (1998); Jung et al., Eur J Immunol lymphoma, melanoma, and/or as described 25(8): 2413-2416 (1995); Mori et al., J below under “Hyperproliferative Disorders”). Allergy Clin Immunol 106(1 Pt 2): 558-564 Preferred indications include neoplasms and (2000); and Koning et al., Cytokine cancers, such as, leukemia, lymphoma, 9(6): 427-436 (1997), the contents of each of melanoma, and prostate, breast, lung, colon, which are herein incorporated by reference pancreatic, esophageal, stomach, brain, liver in its entirety. Human T cells that may be and urinary cancer. Other preferred indications used according to these assays may be include benign dysproliferative disorders and isolated using techniques disclosed herein pre-neoplastic conditions, such as, for example, or otherwise known in the art. Human T hyperplasia, metaplasia, and/or dysplasia. cells are primary human lymphocytes that Preferred indications include anemia, mature in the thymus and express a T cell pancytopenia, leukopenia, thrombocytopenia, receptor and CD3, CD4, or CD8. These leukemias, Hodgkin's disease, acute cells mediate humoral or cell-mediated lymphocytic anemia (ALL), plasmacytomas, immunity and may be preactivated to multiple myeloma, Burkitt's lymphoma, enhance responsiveness to arthritis, AIDS, granulomatous disease, immunomodulatory factors. inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, and Lyme Disease. 98 Production of IL-6 IL-6 FMAT. IL-6 is produced by T cells A highly preferred embodiment of the invention and has strong effects on B cells. IL-6 includes a method for stimulating (e.g., participates in IL-4 induced IgE production increasing) IL-6 production. An alternative and increases IgA production (IgA plays a highly preferred embodiment of the invention role in mucosal immunity). IL-6 induces includes a method for inhibiting (e.g., reducing) cytotoxic T cells. Deregulated expression IL-6 production. A highly preferrred indication of IL-6 has been linked to autoimmune is the stimulation or enhancement of mucosal disease, plasmacytomas, myelomas, and immunity. Highly preferred indications chronic hyperproliferative diseases. Assays include blood disorders (e.g., as described for immunomodulatory and differentiation below under “Immune Activity”, “Blood- factor proteins produced by a large variety Related Disorders”, and/or ““Cardiovascular of cells where the expression level is Disorders””), and infection (e.g., as described strongly regulated by cytokines, growth below under “Infectious Disease”). Highly factors, and hormones are well known in the preferred indications include autoimmune art and may be used or routinely modified to diseases (e.g., rheumatoid arthritis, systemic assess the ability of polypeptides of the lupus erythematosis, multiple sclerosis and/or invention (including antibodies and agonists as described below) and immunodeficiencies or antagonists of the invention) to mediate (e.g., as described below). Highly preferred immunomodulation and differentiation and indications also include boosting a B cell- modulate T cell proliferation and function. mediated immune response and alternatively Exemplary assays that test for suppressing a B cell-mediated immune immunomodulatory proteins evaluate the response. Highly preferred indications include production of cytokines, such as IL-6, and inflammation and inflammatory the stimulation and upregulation of T cell disorders. Additional highly preferred proliferation and functional activities. Such indications include asthma and allergy. assays that may be used or routinely Highly preferred indications include neoplastic modified to test immunomodulatory and diseases (e.g., myeloma, plasmacytoma, diffferentiation activity of polypeptides of leukemia, lymphoma, melanoma, and/or as the invention (including antibodies and described below under “Hyperproliferative agonists or antagonists of the invention) Disorders”). Highly preferred indications include assays disclosed in Miraglia et al., J include neoplasms and cancers, such as, Biomolecular Screening 4: 193-204(1999); myeloma, plasmacytoma, leukemia, lymphoma, Rowland et al., ““Lymphocytes: a practical melanoma, and prostate, breast, lung, colon, approach”” Chapter 6: 138-160 (2000); and pancreatic, esophageal, stomach, brain, liver Verhasselt et al., J Immunol 158: 2919-2925 and urinary cancer. Other preferred indications (1997), the contents of each of which are include benign dysproliferative disorders and herein incorporated by reference in its pre-neoplastic conditions, such as, for example, entirety. Human dendritic cells that may be hyperplasia, metaplasia, and/or dysplasia. used according to these assays may be Preferred indications include anemia, isolated using techniques disclosed herein pancytopenia, leukopenia, thrombocytopenia, or otherwise known in the art. Human Hodgkin's disease, acute lymphocytic anemia dendritic cells are antigen presenting cells in (ALL), multiple myeloma, Burkitt's lymphoma, suspension culture, which, when activated arthritis, AIDS, granulomatous disease, by antigen and/or cytokines, initiate and inflammatory bowel disease, sepsis, upregulate T cell proliferation and neutropenia, neutrophilia, psoriasis, suppression functional activities. of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, and Lyme Disease. An additonal preferred indication is infection (e.g., an infectious disease as described below under ““Infectious Disease””). 99 Production of IL-8 by Assays measuring production of IL-8 are Highly preferred indications include by endothelial cells well known in the art and may be used or immunological and inflammatory disorders (such as Human routinely modified to assess the ability of (e.g., such as allergy, asthma, leukemia, etc. and Umbilical Cord polypeptides of the invention (including as described below under “Immune Activity”, Endothelial Cells). antibodies and agonists or antagonists of the and “Blood-Related Disorders”). Highly invention) to regulate production and/or preferred indications also includie autoimmune secretion of IL-8. For example, FMAT may disorders (e.g., rheumatoid arthritis, systemic be used or routinely modified to assess the lupus erythematosis, Crohn″s disease, multiple ability of polypeptides of the invention sclerosis and/or as described below), neoplastic (including antibodies and agonists or disorders (e.g., organ cancers such as lung, antagonists of the invention) to regulate liver, colon cancer, and/or as described below production and/or secretion of IL-8 from under “Hyperproliferative Disorders”), and endothelial cells (such as human umbilical cardiovascular disorders (e.g. such as described vein endothelial cells (HUVEC)). HUVECs below under “Cardiovascular Disorders”). are endothelial cells which line venous Preferred indications include thrombosis, blood vessels, and are involved in functions bacteremia and sepsis syndrome and that include, but are not limited to, consequent complications (such as acute angiogenesis, vascular permeability, respiratory distress syndrome and systemic vascular tone, and immune cell ischemia-reperfusion resulting from septic extravasation. Endothelial cells play a shock), restnosis and atherosclerosis. pivotal role in the initiation and perpetuation of inflammation and secretion of IL-8 may play an important role in recruitment and activation of immune cells such as neutrophils, macrophages, and lymphocytes. 100 Production of IL-8 by Assays measuring production of IL-8 are endothelial cells well known in the art and may be used or (such as Human routinely modified to assess the ability of Umbilical Cord polypeptides of the invention (including Endothelial Cells). antibodies and agonists or antagonists of the invention) to regulate production and/or secretion of IL-8. For example, FMAT may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate production and/or secretion of IL-8 from endothelial cells (such as human umbilical vein endothelial cells (HUVEC)). HUVECs are endothelial cells which line venous blood vessels, and are involved in functions that include, but are not limited to, angiogenesis, vascular permeability, vascular tone, and immune cell extravasation. Endothelial cells play a pivotal role in the initiation and perpetuation of inflammation and secretion of IL-8 may play an important role in recruitment and activation of immune cells such as neutrophils, macrophages, and lymphocytes. 101 Production of IL-8 by Assay that measures the production of the immune cells (such chemokine interleukin-8 (IL-8) from as the human EOL-1 immune cells (such as the EOL-1 human eosinophil cells) eosinophil cell line) are well known in the art (for example, measurement of IL-8 production by FMAT) and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to promote or inhibit. Eosinophils are a type of immune cell important in allergic responses; they are recruited to tissues and mediate the inflammatory response of late stage allergic reaction. IL8 is a strong immunomodulator and may have a potential proinflammatory role in immunological diseases and disorders (such as allergy and asthma). 102 Production of IL6 by Assay to measure regulation of production primary human aortic of Interleukin-6 (IL-6) by either human smooth muscle or aortic smooth muscle cells or normal human normal human dermal dermal fibroblasts minus or plus fibroblast cells costimulation with TNFalpha (TNFa). (without or with Human aortic smooth muscle cells or costimulation with normal human dermal fibroblasts may be TNFalpha). obtained from commercial sources; these cells are important structural and functional components of blood vessels and connective tissue, respectively. Interleukin-6 (IL-6) is a key molecule in chronic inflammation and has been implicated in the progression of arteriosclerosis, stroke, arthritis and other vascular and inflammatory diseases. Deregulated expression of IL-6 has been linked to autoimmune disease, plasmacytomas, myelomas, and chronic hyperproliferative diseases. Assays for immunomodulatory and differentiation factor proteins produced by a large variety of cells where the expression level is strongly regulated by cytokines, growth factors, and hormones are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation and production of IL-6. 103 Production of MCP-1 MCP-1 FMAT. Assays for A highly preferred embodiment of the invention immunomodulatory proteins that are includes a method for stimulating (e.g., produced by a large variety of cells and act increasing) MCP-1 production. An alternative to induce chemotaxis and activation of highly preferred embodiment of the invention monocytes and T cells are well known in includes a method for inhibiting (e.g., reducing) the art and may be used or routinely MCP-1 production. A highly preferred modified to assess the ability of indication is infection (e.g., an infectious polypeptides of the invention (including disease as described below under “Infectious antibodies and agonists or antagonists of the Disease”). Additional highly preferred invention) to mediate immunomodulation, indications include inflammation and induce chemotaxis, and modulate immune inflammatory disorders. Preferred cell activation. Exemplary assays that test indications include blood disorders (e.g., as for immunomodulatory proteins evaluate described below under “Immune Activity”, the production of cell surface markers, such “Blood-Related Disorders”, and/or as monocyte chemoattractant protein ““Cardiovascular Disorders””). Highly preferred (MCP), and the activation of monocytes and indications include autoimmune diseases (e.g., T cells. Such assays that may be used or rheumatoid arthritis, systemic lupus routinely modified to test erythematosis, multiple sclerosis and/or as immunomodulatory and diffferentiation described below) and immunodeficiencies (e.g., activity of polypeptides of the invention as described below). Preferred indications (including antibodies and agonists or also include anemia, pancytopenia, leukopenia, antagonists of the invention) include assays thrombocytopenia, Hodgkin's disease, acute disclosed in Miraglia et al., J Biomolecular lymphocytic anemia (ALL), plasmacytomas, Screening 4: 193-204(1999); Rowland et al., multiple myeloma, Burkitt's lymphoma, ““Lymphocytes: a practical approach”” arthritis, AIDS, granulomatous disease, Chapter 6: 138-160 (2000); Satthaporn and inflammatory bowel disease, sepsis, Eremin, J R Coll Surg Ednb 45(1): 9-19 neutropenia, neutrophilia, psoriasis, suppression (2001); and Verhasselt et al., J Immunol of immune reactions to transplanted organs and 158: 2919-2925 (1997), the contents of each tissues, hemophilia, hypercoagulation, diabetes of which are herein incorporated by mellitus, endocarditis, meningitis (bacterial and reference in its entirety. Human dendritic viral), Lyme Disease, asthma, and allergy cells that may be used according to these Preferred indications also include neoplastic assays may be isolated using techniques diseases (e.g., leukemia, lymphoma, and/or as disclosed herein or otherwise known in the described below under “Hyperproliferative art. Human dendritic cells are antigen Disorders”). Highly preferred indications presenting cells in suspension culture, include neoplasms and cancers, such as, which, when activated by antigen and/or leukemia, lymphoma, prostate, breast, lung, cytokines, initiate and upregulate T cell colon, pancreatic, esophageal, stomach, brain, proliferation and functional activities. liver, and urinary cancer. Other preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. 104 Production of MIP-1alpha FMAT. Assays for A highly preferred embodiment of the invention MIP1alpha immunomodulatory proteins produced by includes a method for stimulating MIP1a activated dendritic cells that upregulate production. An alternative highly preferred monocyte/macrophage and T cell embodiment of the invention includes a method chemotaxis are well known in the art and for inhibiting (e.g., reducing) MIP1a may be used or routinely modified to assess production. A highly preferred indication is the ability of polypeptides of the invention infection (e.g., an infectious disease as (including antibodies and agonists or described below under “Infectious Disease”). antagonists of the invention) to mediate Preferred indications include blood disorders immunomodulation, modulate chemotaxis, (e.g., as described below under “Immune and modulate T cell differentiation. Activity”, “Blood-Related Disorders”, and/or Exemplary assays that test for ““Cardiovascular Disorders””). Highly preferred immunomodulatory proteins evaluate the indications include autoimmune diseases (e.g., production of chemokines, such as rheumatoid arthritis, systemic lupus macrophage inflammatory protein 1 alpha erythematosis, multiple sclerosis and/or as (MIP-1a), and the activation of described below) and immunodeficiencies (e.g., monocytes/macrophages and T cells. Such as described below). Additional highly assays that may be used or routinely preferred indications include inflammation and modified to test immunomodulatory and inflammatory disorders. Preferred indications chemotaxis activity of polypeptides of the also include anemia, pancytopenia, leukopenia, invention (including antibodies and agonists thrombocytopenia, Hodgkin's disease, acute or antagonists of the invention) include lymphocytic anemia (ALL), plasmacytomas, assays disclosed in Miraglia et al., J multiple myeloma, Burkitt's lymphoma, Biomolecular Screening 4: 193-204(1999); arthritis, AIDS, granulomatous disease, Rowland et al., ““Lymphocytes: a practical inflammatory bowel disease, sepsis, approach”” Chapter 6: 138-160 (2000); neutropenia, neutrophilia, psoriasis, suppression Satthaporn and Eremin, J R Coll Surg Ednb of immune reactions to transplanted organs and 45(1): 9-19 (2001); Drakes et al., Transp tissues, hemophilia, hypercoagulation, diabetes Immunol 8(1): 17-29 (2000); Verhasselt et mellitus, endocarditis, meningitis, Lyme al., J Immunol 158: 2919-2925 (1997); and Disease, asthma, and allergy. Preferred Nardelli et al., J Leukoc Biol 65: 822-828 indications also include neoplastic diseases (1999), the contents of each of which are (e.g., leukemia, lymphoma, and/or as described herein incorporated by reference in its below under “Hyperproliferative Disorders”). entirety. Human dendritic cells that may be Highly preferred indications include neoplasms used according to these assays may be and cancers, such as, leukemia, lymphoma, isolated using techniques disclosed herein prostate, breast, lung, colon, pancreatic, or otherwise known in the art. Human esophageal, stomach, brain, liver, and urinary dendritic cells are antigen presenting cells in cancer. Other preferred indications include suspension culture, which, when activated benign dysproliferative disorders and pre- by antigen and/or cytokines, initiate and neoplastic conditions, such as, for example, upregulate T cell proliferation and hyperplasia, metaplasia, and/or dysplasia. functional activities. 105 Production of RANTES FMAT. Assays for RANTES immunomodulatory proteins that induce chemotaxis of T cells, monocytes, and eosinophils are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, induce chemotaxis, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as RANTES, and the induction of chemotactic responses in immune cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000): Cocchi et al., Science 270(5243): 1811-1815 (1995); and Robinson et al., Clin Exp Immunol 101(3): 398-407 (1995), the contents of each of which are herein incorporated by reference in its entirety. Human immune cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. 106 Production of RANTES FMAT. Assays for RANTES in immunomodulatory proteins that induce bronchial epithelium chemotaxis of T cells, monocytes, and cells eosinophils are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, induce chemotaxis, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as RANTES, and the induction of chemotactic responses in immune cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000): Cocchi et al., Science 270(5243): 1811-1815 (1995); and Robinson et al., Clin Exp Immunol 101(3): 398-407 (1995), the contents of each of which are herein incorporated by reference in its entirety. Epithelial cells were isolated from bronchia/trachea immediately postmortem from humans who were free of known respiratory diseases. See Wu et al., Am Rev Respir Dis. 132(2): 311-20 (1985), the contents of which are herein incorporated by reference in its entirety. 107 Production of RANTES FMAT. Assays for RANTES in immunomodulatory proteins that induce endothelial cells chemotaxis of T cells, monocytes, and (such as human eosinophils are well known in the art and umbilical vein may be used or routinely modified to assess endothelial cells the ability of polypeptides of the invention (HUVEC)) (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, induce chemotaxis, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines, such as RANTES, and the induction of chemotactic responses in immune cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000): Cocchi et al., Science 270(5243): 1811-1815 (1995); and Robinson et al., Clin Exp Immunol 101(3): 398-407 (1995), the contents of each of which are herein incorporated by reference in its entirety. Endothelial cells that may be used according to these assays are publicly available (e.g., through the ATCC ™). Exemplary endothelial cells that may be used according to these assays include human umbilical vein endothelial cells (HUVEC), which are endothelial cells which line venous blood vessels, and are involved in functions that include, but are not limited to, angiogenesis, vascular permeability, vascular tone, and immune cell extravasation. 108 Production of TNF TNFa FMAT. Assays for A highly preferred embodiment of the invention alpha by dendritic immunomodulatory proteins produced by includes a method for inhibiting (e.g., cells activated macrophages, T cells, fibroblasts, decreasing) TNF alpha production. An smooth muscle, and other cell types that alternative highly preferred embodiment of the exert a wide variety of inflammatory and invention includes a method for stimulating cytotoxic effects on a variety of cells are (e.g., increasing) TNF alpha production. well known in the art and may be used or Highly preferred indications include blood routinely modified to assess the ability of disorders (e.g., as described below under polypeptides of the invention (including “Immune Activity”, “Blood-Related antibodies and agonists or antagonists of the Disorders”, and/or ““Cardiovascular invention) to mediate immunomodulation, Disorders””), Highly preferred indications modulate inflammation and cytotoxicity. include autoimmune diseases (e.g., rheumatoid Exemplary assays that test for arthritis, systemic lupus erythematosis, Crohn's immunomodulatory proteins evaluate the disease, multiple sclerosis and/or as described production of cytokines such as tumor below), immunodeficiencies (e.g., as described necrosis factor alpha (TNFa), and the below), boosting a T cell-mediated immune induction or inhibition of an inflammatory response, and suppressing a T cell-mediated or cytotoxic response. Such assays that may immune response. Additional highly preferred be used or routinely modified to test indications include inflammation and immunomodulatory activity of polypeptides inflammatory disorders, and treating joint of the invention (including antibodies and damage in patients with rheumatoid arthritis. agonists or antagonists of the invention) An additional highly preferred indication is include assays disclosed in Miraglia et al., J sepsis. Highly preferred indications include Biomolecular Screening 4: 193-204(1999); neoplastic diseases (e.g., leukemia, lymphoma, Rowland et al., ““Lymphocytes: a practical and/or as described below under approach”” Chapter 6: 138-160 (2000); “Hyperproliferative Disorders”). Additionally, Verhasselt et al., Eur J Immunol highly preferred indications include neoplasms 28(11): 3886-3890 (1198); Dahlen et al., J and cancers, such as, leukemia, lymphoma, Immunol 160(7): 3585-3593 (1998); melanoma, glioma (e.g., malignant glioma), Verhasselt et al., J Immunol 158: 2919-2925 solid tumors, and prostate, breast, lung, colon, (1997); and Nardelli et al., J Leukoc Biol pancreatic, esophageal, stomach, brain, liver 65: 822-828 (1999), the contents of each of and urinary cancer. Other preferred indications which are herein incorporated by reference include benign dysproliferative disorders and in its entirety. Human dendritic cells that pre-neoplastic conditions, such as, for example, may be used according to these assays may hyperplasia, metaplasia, and/or dysplasia. be isolated using techniques disclosed Preferred indications include anemia, herein or otherwise known in the art. pancytopenia, leukopenia, thrombocytopenia, Human dendritic cells are antigen Hodgkin's disease, acute lymphocytic anemia presenting cells in suspension culture, (ALL), plasmacytomas, multiple myeloma, which, when activated by antigen and/or Burkitt's lymphoma, arthritis, AIDS, cytokines, initiate and upregulate T cell granulomatous disease, inflammatory bowel proliferation and functional activities. disease, neutropenia, neutrophilia, psoriasis, suppression of immune reactions to transplanted organs and tissues, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, cardiac reperfusion injury, and asthma and allergy. An additional preferred indication is infection (e.g., an infectious disease as described below under “Infectious Disease”). 109 Production of TNF TNFa FMAT. Assays for alpha by T cells immunomodulatory proteins produced by activated macrophages, T cells, fibroblasts, smooth muscle, and other cell types that exert a wide variety of inflammatory and cytotoxic effects on a variety of cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to mediate immunomodulation, modulate inflammation and cytotoxicity, and mediate humoral and/or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the production of cytokines such as tumor necrosis factor alpha (TNFa), and the induction or inhibition of an inflammatory or cytotoxic response. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Verhasselt et al., Eur J Immunol 28(11): 3886-3890 (1198); Dahlen et al., J Immunol 160(7): 3585-3593 (1998); Verhasselt et al., J Immunol 158: 2919-2925 (1997); and Nardelli et al., J Leukoc Biol 65: 822-828 (1999), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 110 Production of VCAM Assays for measuring expression of VCAM Highly preferred indications include in endothelial cells are well-known in the art and may be used inflammation (acute and chronic), restnosis, (such as human or routinely modified to assess the ability of atherosclerosis, asthma and allergy. Highly umbilical vein polypeptides of the invention (including preferred indications include inflammation and endothelial cells antibodies and agonists or antagonists of the inflammatory disorders, immunological (HUVEC)) invention) to regulate VCAM expression. disorders, neoplastic disorders (e.g. For example, FMAT may be used to meaure cancer/tumorigenesis), and cardiovascular the upregulation of cell surface VCAM-1 disorders (such as described below under expresssion in endothelial cells. Endothelial “Immune Activity”, “Blood-Related cells are cells that line blood vessels, and Disorders”, “Hyperproliferative Disorders” are involved in functions that include, but and/or “Cardiovascular Disorders”). Highly are not limited to, angiogenesis, vascular preferred indications include neoplasms and permeability, vascular tone, and immune cancers such as, for example, leukemia, cell extravasation. Exemplary endothelial lymphoma, melanoma, renal cell carcinoma, cells that may be used according to these and prostate, breast, lung, colon, pancreatic, assays include human umbilical vein esophageal, stomach, brain, liver and urinary endothelial cells (HUVEC), which are cancer. Other preferred indications include available from commercial sources. The benign dysproliferative disorders and pre- expression of VCAM (CD106), a neoplastic conditions, such as, for example, membrane-associated protein, can be hyperplasia, metaplasia, and/or dysplasia. upregulated by cytokines or other factors, and contributes to the extravasation of lymphocytes, leucocytes and other immune cells from blood vessels; thus VCAM expression plays a role in promoting immune and inflammatory responses. 111 Proliferation of Assays for the regulation (i.e. increases or Highly preferred indications include asthma, immune cells (such decreases) of viability and proliferation of allergy, mastocytosis (a rare, heterogeneous as the HMC-1 human cells in vitro are well-known in the art and disorder characterized by excessive mast cell line) may be used or routinely modified to assess accumulation of mast cells, and their the ability of polypeptides of the invention proliferation and action in the skin, central (including antibodies and agonists or nervous system, and other organs). Preferred antagonists of the invention) to regulate indications also include hematopoietic and viability and proliferation of eosinophil immunological disorders (e.g., as described cells and cell lines. For example, the below under “Immune Activity”, and “Blood- CellTiter-Gloô Luminescent Cell Viability Related Disorders”), infection (e.g., as Assay (Promega Corp., Madison, WI, USA) described below under “Infectious Disease”), can be used to measure the number of autoimmune diseases (e.g., rheumatoid arthritis, viable cells in culture based on quantitation systemic lupus erythematosis, multiple sclerosis of the ATP present which signals the and/or as described below), and presence of metabolically active cells. immunodeficiencies (e.g., as described below). Mast cells are found in connective and mucosal tissues throughout the body. Mast cell activation (via immunoglobulin E- antigen, promoted by T helper cell type 2 cytokines) is an important component of allergic disease. Dysregulation of mast cell apoptosis may play a role in allergic disease and mast cell tumor survival. Mast cell lines that may be used according to these assays are publicly available and/or may be routinely generated. Exemplary mast cells that may be used according to these assays include HMC-1, which is an immature human mast cell line established from the peripheral blood of a patient with mast cell leukemia, and exhibits many characteristics of immature mast cells. 112 Proliferation of pre- Assays for the regulation (i.e. increases or adipose cells (such as decreases) of viability and proliferation of 3T3-L1 cells) cells in vitro are well-known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to regulate viability and proliferation of pre-adipose cells and cell lines. For example, the CellTiter-Gloô Luminescent Cell Viability Assay (Promega Corp., Madison, WI, USA) can be used to measure the number of viable cells in culture based on quantitation of the ATP present which signals the presence of metabolically active cells. 3T3-L1 is a mouse preadipocyte cell line. It is a continuous substrain of 3T3 fibroblast cells developed through clonal isolation. Cells were differentiated to an adipose-like state before being used in the screen. See Green H and Meuth M., Cell 3: 127-133 (1974), which is herein incorporated by reference in its entirety. 113 Protection from Caspase Apoptosis Rescue. Assays for A highly preferred embodiment of the invention Endothelial Cell caspase apoptosis rescue are well known in includes a method for stimulating endothelial Apoptosis. the art and may be used or routinely cell growth. An alternative highly preferred modified to assess the ability of the embodiment of the invention includes a method polypeptides of the invention (including for inhibiting endothelial cell growth. A antibodies and agonists or antagonists of the highly preferred embodiment of the invention invention) to inhibit caspase protease- includes a method for stimulating endothelial mediated apoptosis. Exemplary assays for cell proliferation. An alternative highly caspase apoptosis that may be used or preferred embodiment of the invention includes routinely modified to test caspase apoptosis a method for inhibiting endothelial cell rescue of polypeptides of the invention proliferation. A highly preferred (including antibodies and agonists or embodiment of the invention includes a method antagonists of the invention) include the for stimulating endothelial cell growth. An assays disclosed in Romeo et al., alternative highly preferred embodiment of the Cardiovasc Res 45(3): 788-794 (2000); invention includes a method for inhibiting Messmer et al., Br J Pharmacol 127(7): endothelial cell growth. A highly preferred 1633-1640 (1999); and J Atheroscler embodiment of the invention includes a method Thromb 3(2): 75-80 (1996); the contents of for stimulating apoptosis of endothelial cells. each of which are herein incorporated by An alternative highly preferred embodiment of reference in its entirety. Endothelial cells the invention includes a method for inhibiting that may be used according to these assays (e.g., decreasing) apoptosis of endothelial cells. are publicly available (e.g., through A highly preferred embodiment of the invention commercial sources). Exemplary includes a method for stimulating angiogenisis. endothelial cells that may be used according An alternative highly preferred embodiment of to these assays include bovine aortic the invention includes a method for inhibiting endothelial cells (bAEC), which are an angiogenesis. A highly preferred example of endothelial cells which line embodiment of the invention includes a method blood vessels and are involved in functions for reducing cardiac hypertrophy. An that include, but are not limited to, alternative highly preferred embodiment of the angiogenesis, vascular permeability, invention includes a method for inducing vascular tone, and immune cell cardiac hypertrophy. Highly preferred extravasation. indications include neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., heart disease, congestive heart failure, hypertension, aortic stenosis, cardiomyopathy, valvular regurgitation, left ventricular dysfunction, atherosclerosis and atherosclerotic vascular disease, diabetic nephropathy, intracardiac shunt, cardiac hypertrophy, myocardial infarction, chronic hemodynamic overload, and/or as described below under “Cardiovascular Disorders”). Highly preferred indications include cardiovascular, endothelial and/or angiogenic disorders (e.g., systemic disorders that affect vessels such as diabetes mellitus, as well as diseases of the vessels themselves, such as of the arteries, capillaries, veins and/or lymphatics). Highly preferred are indications that stimulate angiogenesis and/or cardiovascularization. Highly preferred are indications that inhibit angiogenesis and/or cardiovascularization. Highly preferred indications include antiangiogenic activity to treat solid tumors, leukemias, and Kaposi″s sarcoma, and retinal disorders. Highly preferred indications include neoplasms and cancer, such as, Kaposi″s sarcoma, hemangioma (capillary and cavernous), glomus tumors, telangiectasia, bacillary angiomatosis, hemangioendothelioma, angiosarcoma, haemangiopericytoma, lymphangioma, lymphangiosarcoma. Highly preferred indications also include cancers such as, prostate, breast, lung, colon, pancreatic, esophageal, stomach, brain, liver, and urinary cancer. Preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. Highly preferred indications also include arterial disease, such as, atherosclerosis, hypertension, coronary artery disease, inflammatory vasculitides, Reynaud″s disease and Reynaud″s phenomenom, aneurysms, restenosis; venous and lymphatic disorders such as thrombophlebitis, lymphangitis, and lymphedema; and other vascular disorders such as peripheral vascular disease, and cancer. Highly preferred indications also include trauma such as wounds, burns, and injured tissue (e.g., vascular injury such as, injury resulting from balloon angioplasty, and atheroschlerotic lesions), implant fixation, scarring, ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular disease, renal diseases such as acute renal failure, and osteoporosis. Additional highly preferred indications include stroke, graft rejection, diabetic or other retinopathies, thrombotic and coagulative disorders, vascularitis, lymph angiogenesis, sexual disorders, age-related macular degeneration, and treatment/ prevention of endometriosis and related conditions. Additional highly preferred indications include fibromas, heart disease, cardiac arrest, heart valve disease, and vascular disease. Preferred indications include blood disorders (e.g., as described below under “Immune Activity”, “Blood-Related Disorders”, and/or “Cardiovascular Disorders”). Preferred indications include autoimmune diseases (e.g., rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis and/or as described below) and immunodeficiencies (e.g., as described below). Additional preferred indications include inflammation and inflammatory disorders (such as acute and chronic inflammatory diseases, e.g., inflammatory bowel disease and Crohn's disease), and pain management. 114 Regulation The mixed lymphocyte reaction assay (inhibition or (MLR) (see e.g., Example: “Detection of activation) of Inhibition of a Mixed Lymphocyte immune cell Reaction” below) is a complex in vitro proliferation. assay of T-cell responsiveness and immune cell activation. This assay is useful, for example, as an in vitro model of allograft rejection and graft versus host disease. In this assays PBMCs from human donors are mixed, cultured, and monitored for thymidine incorporation (a measure of cell proliferation) to identify polypeptides of the invention (including antibodies and agonists or antagonists of the invention) that may activate or inhibit immune responses. 115 Regulation of Caspase Apoptosis. Assays for caspase A highly preferred indication is diabetes apoptosis in apoptosis are well known in the art and may mellitus. An additional highly preferred pancreatic beta cells. be used or routinely modified to assess the indication is a complication associated with ability of polypeptides of the invention diabetes (e.g., diabetic retinopathy, diabetic (including antibodies and agonists or nephropathy, kidney disease (e.g., renal failure, antagonists of the invention) to promote nephropathy and/or other diseases and disorders caspase protease-mediated apoptosis. as described in the ““Renal Disorders”” section Apoptosis in pancreatic beta is associated below), diabetic neuropathy, nerve disease and with induction and progression of diabetes. nerve damage (e.g., due to diabetic neuropathy), Exemplary assays for caspase apoptosis that blood vessel blockage, heart disease, stroke, may be used or routinely modified to test impotence (e.g., due to diabetic neuropathy or capase apoptosis activity of polypeptides of blood vessel blockage), seizures, mental the invention (including antibodies and confusion, drowsiness, nonketotic agonists or antagonists of the invention) hyperglycemic-hyperosmolar coma, include the assays disclosed in: Loweth, AC, cardiovascular disease (e.g., heart disease, et al., FEBS Lett, 400(3): 285-8 (1997); atherosclerosis, microvascular disease, Saini, KS, et al., Biochem Mol Biol Int, hypertension, stroke, and other diseases and 39(6): 1229-36 (1996); Krautheim, A., et al., disorders as described in the ““Cardiovascular Br J Pharmacol, 129(4): 687-94 (2000); Disorders”” section below), dyslipidemia, Chandra J, et al., Diabetes, 50 Suppl 1: S44-7 endocrine disorders (as described in the (2001); Suk K, et al., J Immunol, ““Endocrine Disorders”” section below), 166(7): 4481-9 (2001); Tejedo J, et al., neuropathy, vision impairment (e.g., diabetic FEBS Lett, 459(2): 238-43 (1999); Zhang, S., retinopathy and blindness), ulcers and impaired et al., FEBS Lett, 455(3): 315-20 (1999); wound healing, and infection (e.g., infectious Lee et al., FEBS Lett 485(2-3): 122-126 diseases and disorders as described in the (2000); Nor et al., J Vasc Res 37(3): 209-218 ““Infectious Diseases”” section below, (2000); and Karsan and Harlan, J especially of the urinary tract and skin), carpal Atheroscler Thromb 3(2): 75-80 (1996); the tunnel syndrome and Dupuytren's contracture). contents of each of which are herein An additional highly preferred indication is incorporated by reference in its entirety. obesity and/or complications associated with Pancreatic cells that may be used according obesity. Additional highly preferred indications to these assays are publicly available (e.g., include weight loss or alternatively, weight through the ATCC ™) and/or may be gain. Aditional highly preferred routinely generated. Exemplary pancreatic indications are complications associated with cells that may be used according to these insulin resistance. assays include RIN-m. RIN-m is a rat adherent pancreatic beta cell insulinoma cell line derived from a radiation induced transplantable rat islet cell tumor. The cells produce and secrete islet polypeptide hormones, and produce insulin, somatostatin, and possibly glucagon. ATTC: #CRL-2057 Chick et al. Proc. Natl. Acad. Sci. 1977 74: 628; AF et al. Proc. Natl. Acad. Sci. 1980 77: 3519. 116 Regulation of Caspase Apoptosis. Assays for caspase Preferred embodiments of the invention include apoptosis of immune apoptosis are well known in the art and may using polypeptides of the invention (or cells (such as mast be used or routinely modified to assess the antibodies, agonists, or antagonists thereof) in cells). ability of polypeptides of the invention detection, diagnosis, prevention, and/or (including antibodies and agonists or treatment of asthma, allergy, hypersensitivity antagonists of the invention) to regulate and inflammation. caspase protease-mediated apoptosis in immune cells (such as, for example, in mast cells). Mast cells are found in connective and mucosal tissues throughout the body, and their activation via immunoglobulin E- antigen, promoted by T helper cell type 2 cytokines, is an important component of allergic disease. Dysregulation of mast cell apoptosis may play a role in allergic disease and mast cell tumor survival. Exemplary assays for caspase apoptosis that may be used or routinely modified to test capase apoptosis activity induced by polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Masuda A, et al., J Biol Chem, 276(28): 26107-26113 (2001); Yeatman CF 2nd, et al., J Exp Med, 192(8): 1093-1103 (2000); Lee et al., FEBS Lett 485(2-3): 122-126 (2000); Nor et al., J Vasc Res 37(3): 209-218 (2000); and Karsan and Harlan, J Atheroscler Thromb 3(2): 75-80 (1996); the contents of each of which are herein incorporated by reference in its entirety. Immune cells that may be used according to these assays are publicly available (e.g., through commercial sources). Exemplary immune cells that may be used according to these assays include mast cells such as the HMC human mast cell line. 117 Regulation of Kinase assays, for example an Elk-1 kinase Preferred embodiments of the invention include proliferation and/or assay for ERK signal transduction that using polypeptides of the invention (or differentiation in regulates cell proliferation or antibodies, agonists, or antagonists thereof) in immune cells (such differentiation, are well known in the art detection, diagnosis, prevention, and/or as mast cells). and may be used or routinely modified to treatment of asthma, allergy, hypersensitivity assess the ability of polypeptides of the and inflammation. invention (including antibodies and agonists or antagonists of the invention) to promote or inhibit cell proliferation, activation, and differentiation. Exemplary assays for ERK kinase activity that may be used or routinely modified to test ERK kinase-induced activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include the assays disclosed in: Ali H, et al., J Immunol, 165(12): 7215-7223 (2000); Tam SY, et al., Blood, 90(5): 1807-1820 (1997); Forrer et al., Biol Chem 379(8-9): 1101-1110 (1998); Berra et al., Biochem Pharmacol 60(8): 1171-1178 (2000); Gupta et al., Exp Cell Res 247(2): 495-504 (1999); Chang and Karin, Nature 410(6824): 37-40 (2001); and Cobb MH, Prog Biophys Mol Biol 71(3-4): 479-500 (1999); the contents of each of which are herein incorporated by reference in its entirety. Exemplary immune cells that may be used according to these assays include human mast cells such as the HMC- 1 cell line. 118 Regulation of Assays for the regulation of transcription of A highly preferred indication is diabetes transcription of Malic Malic Enzyme are well-known in the art mellitus. An additional highly preferred Enzyme in adipocytes and may be used or routinely modified to indication is a complication associated with assess the ability of polypeptides of the diabetes (e.g., diabetic retinopathy, diabetic invention (including antibodies and agonists nephropathy, kidney disease (e.g., renal failure, or antagonists of the invention) to regulate nephropathy and/or other diseases and disorders transcription of Malic Enzyme, a key as described in the “Renal Disorders” section enzyme in lipogenesis. Malic enzyme is below), diabetic neuropathy, nerve disease and involved in lipogenesis and its expression is nerve damage (e.g., due to diabetic neuropathy), stimulted by insulin. ME promoter contains blood vessel blockage, heart disease, stroke, two direct repeat (DR1)-like elements impotence (e.g., due to diabetic neuropathy or MEp and MEd identified as putative PPAR blood vessel blockage), seizures, mental response elements. ME promoter may also confusion, drowsiness, nonketotic responds to AP1 and other transcription hyperglycemic-hyperosmolar coma, factors. Exemplary assays that may be used cardiovascular disease (e.g., heart disease, or routinely modified to test for regulation atherosclerosis, microvascular disease, of transcription of Malic Enzyme (in hypertension, stroke, and other diseases and adipoocytes) by polypeptides of the disorders as described in the “Cardiovascular invention (including antibodies and agonists Disorders” section below), dyslipidemia, or antagonists of the invention) include endocrine disorders (as described in the assays disclosed in: Streeper, R. S., et al., “Endocrine Disorders” section below), Mol Endocrinol, 12(11): 1778-91 (1998); neuropathy, vision impairment (e.g., diabetic Garcia-Jimenez, C., et al., Mol Endocrinol, retinopathy and blindness), ulcers and impaired 8(10): 1361-9 (1994); Barroso, I., et al., J wound healing, and infection (e.g., infectious Biol Chem, 274(25): 17997-8004 (1999); diseases and disorders as described in the Ijpenberg, A., et al., J Biol Chem, “Infectious Diseases” section below, especially 272(32): 20108-20117 (1997); Berger, et al., of the urinary tract and skin), carpal tunnel Gene 66: 1-10 (1988); and, Cullen, B., et al., syndrome and Dupuytren's contracture). Methods in Enzymol. 216: 362-368 (1992), An additional highly preferred indication is the contents of each of which is herein obesity and/or complications associated with incorporated by reference in its entirety. obesity. Additional highly preferred indications Hepatocytes that may be used according to include weight loss or alternatively, weight these assays are publicly available (e.g., gain. Aditional highly preferred through the ATCC ™) and/or may be indications are complications associated with routinely generated. Exemplary insulin resistance. hepatocytes that may be used according to these assays includes the H4IIE rat liver hepatoma cell line. 119 Regulation of Assays for the regulation of transcription of A highly preferred indication is diabetes transcription of Malic Malic Enzyme are well-known in the art mellitus. An additional highly preferred Enzyme in and may be used or routinely modified to indication is a complication associated with hepatocytes assess the ability of polypeptides of the diabetes (e.g., diabetic retinopathy, diabetic invention (including antibodies and agonists nephropathy, kidney disease (e.g., renal failure, or antagonists of the invention) to regulate nephropathy and/or other diseases and disorders transcription of Malic Enzyme, a key as described in the ““Renal Disorders”” section enzyme in lipogenesis. Malic enzyme is below), diabetic neuropathy, nerve disease and involved in lipogenesis and its expression is nerve damage (e.g., due to diabetic neuropathy), stimulted by insulin. ME promoter contains blood vessel blockage, heart disease, stroke, two direct repeat (DR1)-like elements impotence (e.g., due to diabetic neuropathy or MEp and MEd identified as putative PPAR blood vessel blockage), seizures, mental response elements. ME promoter may also confusion, drowsiness, nonketotic responds to AP1 and other transcription hyperglycemic-hyperosmolar coma, factors. Exemplary assays that may be used cardiovascular disease (e.g., heart disease, or routinely modified to test for regulation atherosclerosis, microvascular disease, of transcription of Malic Enzyme (in hypertension, stroke, and other diseases and hepatocytes) by polypeptides of the disorders as described in the ““Cardiovascular invention (including antibodies and agonists Disorders”” section below), dyslipidemia, or antagonists of the invention) include endocrine disorders (as described in the assays disclosed in: Streeper, R. S., et al., ““Endocrine Disorders”” section below), Mol Endocrinol, 12(11): 1778-91 (1998); neuropathy, vision impairment (e.g., diabetic Garcia-Jimenez, C., et al., Mol Endocrinol, retinopathy and blindness), ulcers and impaired 8(10): 1361-9 (1994); Barroso, I., et al., J wound healing, and infection (e.g., infectious Biol Chem, 274(25): 17997-8004 (1999); diseases and disorders as described in the Ijpenberg, A., et al., J Biol Chem, ““Infectious Diseases”” section below, 272(32): 20108-20117 (1997); Berger, et al., especially of the urinary tract and skin), carpal Gene 66: 1-10 (1988); and, Cullen, B., et al., tunnel syndrome and Dupuytren's contracture). Methods in Enzymol. 216: 362-368 (1992), An additional highly preferred indication is the contents of each of which is herein obesity and/or complications associated with incorporated by reference in its entirety. obesity. Additional highly preferred indications Hepatocytes that may be used according to include weight loss or alternatively, weight these assays are publicly available (e.g., gain. Aditional highly preferred through the ATCC ™) and/or may be indications are complications associated with routinely generated. Exemplary insulin resistance. hepatocytes that may be used according to these assays includes the mouse 3T3-L1 cell line. 3T3-L1 is a mouse preadipocyte cell line (adherent). It is a continuous substrain of 3T3 fibroblasts developed through clonal isolation. Cells undergo a pre-adipocyte to adipose-like conversion under appropriate differentiation culture conditions. 120 Regulation of Assays for the regulation of transcription A highly preferred indication is diabetes transcription through through the FAS promoter element are well- mellitus. An additional highly preferred the FAS promoter known in the art and may be used or indication is a complication associated with element in routinely modified to assess the ability of diabetes (e.g., diabetic retinopathy, diabetic hepatocytes polypeptides of the invention (including nephropathy, kidney disease (e.g., renal failure, antibodies and agonists or antagonists of the nephropathy and/or other diseases and disorders invention) to activate the FAS promoter as described in the “Renal Disorders” section element in a reporter construct and to below), diabetic neuropathy, nerve disease and regulate transcription of FAS, a key enzyme nerve damage (e.g., due to diabetic neuropathy), for lipogenesis. FAS promoter is regulated blood vessel blockage, heart disease, stroke, by many transcription factors including impotence (e.g., due to diabetic neuropathy or SREBP. Insulin increases FAS gene blood vessel blockage), seizures, mental transcription in livers of diabetic mice. This confusion, drowsiness, nonketotic stimulation of transcription is also hyperglycemic-hyperosmolar coma, somewhat glucose dependent. Exemplary cardiovascular disease (e.g., heart disease, assays that may be used or routinely atherosclerosis, microvascular disease, modified to test for FAS promoter element hypertension, stroke, and other diseases and activity (in hepatocytes) by polypeptides of disorders as described in the “Cardiovascular the invention (including antibodies and Disorders” section below), dyslipidemia, agonists or antagonists of the invention) endocrine disorders (as described in the include assays disclosed in Xiong, S., et al., “Endocrine Disorders” section below), Proc Natl Acad Sci U.S.A., 97(8): 3948-53 neuropathy, vision impairment (e.g., diabetic (2000); Roder, K., et al., Eur J Biochem, retinopathy and blindness), ulcers and impaired 260(3): 743-51 (1999); Oskouian B, et al., wound healing, and infection (e.g., infectious Biochem J, 317 (Pt 1): 257-65 (1996); diseases and disorders as described in the Berger, et al., Gene 66: 1-10 (1988); and, “Infectious Diseases” section below, especially Cullen, B., et al., Methods in Enzymol. of the urinary tract and skin), carpal tunnel 216: 362-368 (1992), the contents of each of syndrome and Dupuytren's contracture). which is herein incorporated by reference in An additional highly preferred indication is its entirety. Hepatocytes that may be used obesity and/or complications associated with according to these assays, such as H4IIE obesity. Additional highly preferred indications cells, are publicly available (e.g., through include weight loss or alternatively, weight the ATCC ™) and/or may be routinely gain. Aditional highly preferred generated. Exemplary hepatocytes that may indications are complications associated with be used according to these assays include insulin resistance. rat liver hepatoma cell line(s) inducible with glucocorticoids, insulin, or cAMP derivatives. 121 Regulation of Assays for the regulation of transcription A highly preferred indication is diabetes transcription through through the PEPCK promoter are well- mellitus. An additional highly preferred the PEPCK promoter known in the art and may be used or indication is a complication associated with in hepatocytes routinely modified to assess the ability of diabetes (e.g., diabetic retinopathy, diabetic polypeptides of the invention (including nephropathy, kidney disease (e.g., renal failure, antibodies and agonists or antagonists of the nephropathy and/or other diseases and disorders invention) to activate the PEPCK promoter as described in the “Renal Disorders” section in a reporter construct and regulate liver below), diabetic neuropathy, nerve disease and gluconeogenesis. Exemplary assays for nerve damage (e.g., due to diabetic neuropathy), regulation of transcription through the blood vessel blockage, heart disease, stroke, PEPCK promoter that may be used or impotence (e.g., due to diabetic neuropathy or routinely modified to test for PEPCK blood vessel blockage), seizures, mental promoter activity (in hepatocytes) of confusion, drowsiness, nonketotic polypeptides of the invention (including hyperglycemic-hyperosmolar coma, antibodies and agonists or antagonists of the cardiovascular disease (e.g., heart disease, invention) include assays disclosed in atherosclerosis, microvascular disease, Berger et al., Gene 66: 1-10 (1998); Cullen hypertension, stroke, and other diseases and and Malm, Methods in Enzymol 216: 362-368 disorders as described in the “Cardiovascular (1992); Henthorn et al., Proc Natl Acad Disorders” section below), dyslipidemia, Sci USA 85: 6342-6346 (1988); Lochhead et endocrine disorders (as described in the al., Diabetes 49(6): 896-903 (2000); and “Endocrine Disorders” section below), Yeagley et al., J Biol Chem 275(23): 17814-17820 neuropathy, vision impairment (e.g., diabetic (2000), the contents of each of which retinopathy and blindness), ulcers and impaired is herein incorporated by reference in its wound healing, infection (e.g., an infectious entirety. Hepatocyte cells that may be used diseases or disorders as described in the according to these assays are publicly “Infectious Diseases” section below, especially available (e.g., through the ATCC ™) and/or of the urinary tract and skin), carpal tunnel may be routinely generated. Exemplary syndrome and Dupuytren's contracture). liver hepatoma cells that may be used An additional highly preferred indication is according to these assays include H4lle obesity and/or complications associated with cells, which contain a tyrosine amino obesity. Additional highly preferred indications transferase that is inducible with include weight loss or alternatively, weight glucocorticoids, insulin, or cAMP gain. Additional highly preferred derivatives. indications are complications associated with insulin resistance. Additional highly preferred indications are disorders of the musculoskeletal systems including myopathies, muscular dystrophy, and/or as described herein. Additional highly preferred indications include glycogen storage disease (e.g., glycogenoses), hepatitis, gallstones, cirrhosis of the liver, degenerative or necrotic liver disease, alcoholic liver diseases, fibrosis, liver regeneration, metabolic disease, dyslipidemia and cholesterol metabolism, and hepatocarcinomas. Highly preferred indications include blood disorders (e.g., as described below under “Immune Activity”, “Cardiovascular Disorders”, and/or “Blood-Related Disorders”), immune disorders (e.g., as described below under “Immune Activity”), infection (e.g., an infectious disease and/or disorder as described below under “Infectious Disease”), endocrine disorders (e.g., as described below under “Endocrine Disorders”), and neural disorders (e.g., as described below under “Neural Activity and Neurological Diseases”). Additional preferred indications include neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”). Preferred indications include neoplasms and cancers, such as, leukemia, lymphoma, prostate, breast, lung, colon, pancreatic, esophageal, stomach, brain, and urinary cancer. A highly preferred indication is liver cancer. Other preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. 122 Regulation of Assays for the regulation of transcription A highly preferred indication is diabetes transcription via through the DMEF1 response element are mellitus. Additional highly preferred DMEF1 response well-known in the art and may be used or indications include complications associated element in adipocytes routinely modified to assess the ability of with diabetes (e.g., diabetic retinopathy, and pre-adipocytes polypeptides of the invention (including diabetic nephropathy, kidney disease (e.g., renal antibodies and agonists or antagonists of the failure, nephropathy and/or other diseases and invention) to activate the DMEF1 response disorders as described in the “Renal Disorders” element in a reporter construct (such as that section below), diabetic neuropathy, nerve containing the GLUT4 promoter) and to disease and nerve damage (e.g., due to diabetic regulate insulin production. The DMEF1 neuropathy), blood vessel blockage, heart response element is present in the GLUT4 disease, stroke, impotence (e.g., due to diabetic promoter and binds to MEF2 transcription neuropathy or blood vessel blockage), seizures, factor and another transcription factor that is mental confusion, drowsiness, nonketotic required for insulin regulation of Glut4 hyperglycemic-hyperosmolar coma, expression in skeletal muscle. GLUT4 is cardiovascular disease (e.g., heart disease, the primary insulin-responsive glucose atherosclerosis, microvascular disease, transporter in fat and muscle tissue. hypertension, stroke, and other diseases and Exemplary assays that may be used or disorders as described in the “Cardiovascular routinely modified to test for DMEF1 Disorders” section below), dyslipidemia, response element activity (in adipocytes and endocrine disorders (as described in the pre-adipocytes) by polypeptides of the “Endocrine Disorders” section below), invention (including antibodies and agonists neuropathy, vision impairment (e.g., diabetic or antagonists of the invention) include retinopathy and blindness), ulcers and impaired assays disclosed in Thai, M. V., et al., J Biol wound healing, and infection (e.g., infectious Chem, 273(23): 14285-92 (1998); Mora, S., diseases and disorders as described in the et al., J Biol Chem, 275(21): 16323-8 “Infectious Diseases” section below, especially (2000); Liu, M. L., et al., J Biol Chem, of the urinary tract and skin). An additional 269(45): 28514-21 (1994); “Identification of highly preferred indication is obesity and/or a 30-base pair regulatory element and novel complications associated with obesity. DNA binding protein that regulates the Additional highly preferred indications include human GLUT4 promoter in transgenic weight loss or alternatively, weight gain. mice”, J Biol Chem. 2000 Aug Additional highly preferred indications are 4; 275(31): 23666-73; Berger, et al., Gene complications associated with insulin 66: 1-10 (1988); and, Cullen, B., et al., resistance. Methods in Enzymol. 216: 362-368 (1992), the contents of each of which is herein incorporated by reference in its entirety. Adipocytes and pre-adipocytes that may be used according to these assays are publicly available (e.g., through the ATCC ™) and/or may be routinely generated. Exemplary cells that may be used according to these assays include the mouse 3T3-L1 cell line which is an adherent mouse preadipocyte cell line. Mouse 3T3-L1 cells are a continuous substrain of 3T3 fibroblasts developed through clonal isolation. These cells undergo a pre-adipocyte to adipose- like conversion under appropriate differentiation culture conditions. 123 Regulation of Assays for the regulation of viability and A highly preferred indication is diabetes viability and proliferation of cells in vitro are well-known mellitus. An additional highly preferred proliferation of in the art and may be used or routinely indication is a complication associated with pancreatic beta cells. modified to assess the ability of diabetes (e.g., diabetic retinopathy, diabetic polypeptides of the invention (including nephropathy, kidney disease (e.g., renal failure, antibodies and agonists or antagonists of the nephropathy and/or other diseases and disorders invention) to regulate viability and as described in the ““Renal Disorders”” section proliferation of pancreatic beta cells. For below), diabetic neuropathy, nerve disease and example, the Cell Titer-Glo luminescent cell nerve damage (e.g., due to diabetic neuropathy), viability assay measures the number of blood vessel blockage, heart disease, stroke, viable cells in culture based on quantitation impotence (e.g., due to diabetic neuropathy or of the ATP present which signals the blood vessel blockage), seizures, mental presence of metabolically active cells. confusion, drowsiness, nonketotic Exemplary assays that may be used or hyperglycemic-hyperosmolar coma, routinely modified to test regulation of cardiovascular disease (e.g., heart disease, viability and proliferation of pancreatic beta atherosclerosis, microvascular disease, cells by polypeptides of the invention hypertension, stroke, and other diseases and (including antibodies and agonists or disorders as described in the ““Cardiovascular antagonists of the invention) include assays Disorders”” section below), dyslipidemia, disclosed in: Friedrichsen BN, et al., Mol endocrine disorders (as described in the Endocrinol, 15(1): 136-48 (2001); Huotari MA, ““Endocrine Disorders”” section below), et al., Endocrinology, 139(4): 1494-9 neuropathy, vision impairment (e.g., diabetic (1998); Hugl SR, et al., J Biol Chem 1998 retinopathy and blindness), ulcers and impaired Jul 10; 273(28): 17771-9 (1998), the contents wound healing, and infection (e.g., infectious of each of which is herein incorporated by diseases and disorders as described in the reference in its entirety. Pancreatic cells ““Infectious Diseases”” section below, that may be used according to these assays especially of the urinary tract and skin), carpal are publicly available (e.g., through the tunnel syndrome and Dupuytren's contracture). ATCC ™) and/or may be routinely An additional highly preferred indication is generated. Exemplary pancreatic cells that obesity and/or complications associated with may be used according to these assays obesity. Additional highly preferred indications include rat INS-1 cells. INS-1 cells are a include weight loss or alternatively, weight semi-adherent cell line established from gain. Additional highly preferred cells isolated from an X-ray induced rat indications are complications associated with transplantable insulinoma. These cells insulin resistance. retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. Additional exemplary pancreatic cells that may be used according to these assays include HITT15 Cells. HITT15 are an adherent epithelial cell line established from Syrian hamster islet cells transformed with SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 124 Regulation of Assays for the regulation (i.e. increases or Highly preferred indications include viability or decreases) of viability and proliferation of eosinophilia, asthma, allergy, hypersensitivity proliferation of cells in vitro are well-known in the art and reactions, inflammation, and inflammatory immune cells (such may be used or routinely modified to assess disorders. Additional highly preferred as human eosinophil the ability of polypeptides of the invention indications include immune and hematopoietic EOL-1 cells). (including antibodies and agonists or disorders (e.g., as described below under antagonists of the invention) to regulate “Immune Activity”, and “Blood-Related viability and proliferation of eosinophil Disorders”), autoimmune diseases (e.g., cells and cell lines. For example, the rheumatoid arthritis, systemic lupus CellTiter-Gloô Luminescent Cell Viability erythematosis, Crohn″s disease, multiple Assay (Promega Corp., Madison, WI, USA) sclerosis and/or as described below), can be used to measure the number of immunodeficiencies (e.g., as described below). viable cells in culture based on quantitation Highly preferred indications also include of the ATP present which signals the boosting or inhibiting immune cell presence of metabolically active cells. proliferation. Preferred indications include Eosinophils are a type of immune cell neoplastic diseases (e.g., leukemia, lymphoma, important in allergic responses; they are and/or as described below under recruited to tissues and mediate the “Hyperproliferative Disorders”). Highly inflammtory response of late stage allergic preferred indications include boosting an reaction. Eosinophil cell lines that may be eosinophil-mediated immune response, and used according to these assays are publicly suppressing an eosinophil-mediated immune available and/or may be routinely generated. response. Exemplary eosinophil cells that may be used according to these assays include EOL-1 Cells. 125 SEAP in 293/ISRE 126 SEAP in Alk Phos C2C12 127 SEAP in ATP-3T3- L1 128 SEAP in HepG2/Squale- synthetase (stimulation) 129 SEAP in HIB/CRE 130 SEAP in Jurkat-AP1 131 SEAP in Jurkat/IL4 promoter 132 SEAP in Jurkat/IL4 promoter (antiCD3 co-stim) 133 SEAP in Ku812/NFkB (TNF synergy) 134 SEAP in Molt4/SRE 135 SEAP in NK16/STAT6 136 SEAP in OE-21 137 SEAP in OE-33 138 SEAP in SW480 139 SEAP in UMR-106 140 Stimulation of Assays for measuring calcium flux are well- A highly preferred indication is diabetes Calcium Flux in known in the art and may be used or mellitus. An additional highly preferred pancreatic beta cells. routinely modified to assess the ability of indication is a complication associated with polypeptides of the invention (including diabetes (e.g., diabetic retinopathy, diabetic antibodies and agonists or antagonists of the nephropathy, kidney disease (e.g., renal failure, invention) to mobilize calcium. For nephropathy and/or other diseases and disorders example, the FLPR assay may be used to as described in the “Renal Disorders” section measure influx of calcium. Cells normally below), diabetic neuropathy, nerve disease and have very low concentrations of cytosolic nerve damage (e.g., due to diabetic neuropathy), calcium compared to much higher blood vessel blockage, heart disease, stroke, extracellular calcium. Extracellular factors impotence (e.g., due to diabetic neuropathy or can cause an influx of calcium, leading to blood vessel blockage), seizures, mental activation of calcium responsive signaling confusion, drowsiness, nonketotic pathways and alterations in cell functions. hyperglycemic-hyperosmolar coma, Exemplary assays that may be used or cardiovascular disease (e.g., heart disease, routinely modified to measure calcium flux atherosclerosis, microvascular disease, by polypeptides of the invention (including hypertension, stroke, and other diseases and antibodies and agonists or antagonists of the disorders as described in the “Cardiovascular invention) include assays disclosed in: Satin Disorders” section below), dyslipidemia, LS, et al., Endocrinology, 136(10): 4589-601 endocrine disorders (as described in the (1995); Mogami H, et al., Endocrinology, “Endocrine Disorders” section below), 136(7): 2960-6 (1995); Richardson SB, et neuropathy, vision impairment (e.g., diabetic al., Biochem J, 288 (Pt 3): 847-51 (1992); retinopathy and blindness), ulcers and impaired and, Meats, JE, et al., Cell Calcium 1989 wound healing, and infection (e.g., infectious Nov-Dec; 10(8): 535-41 (1989), the contents diseases and disorders as described in the of each of which is herein incorporated by “Infectious Diseases” section below, especially reference in its entirety. Pancreatic cells of the urinary tract and skin), carpal tunnel that may be used according to these assays syndrome and Dupuytren's contracture). are publicly available (e.g., through the An additional highly preferred indication is ATCC ™) and/or may be routinely obesity and/or complications associated with generated. Exemplary pancreatic cells that obesity. Additional highly preferred indications may be used according to these assays include weight loss or alternatively, weight include HITT15 Cells. HITT15 are an gain. Aditional highly preferred adherent epithelial cell line established from indications are complications associated with Syrian hamster islet cells transformed with insulin resistance. SV40. These cells express glucagon, somatostatin, and glucocorticoid receptors. The cells secrete insulin, which is stimulated by glucose and glucagon and suppressed by somatostatin or glucocorticoids. ATTC# CRL-1777 Refs: Lord and Ashcroft. Biochem. J. 219: 547-551; Santerre et al. Proc. Natl. Acad. Sci. USA 78: 4339-4343, 1981. 141 Stimulation of insulin Assays for measuring secretion of insulin A highly preferred indication is diabetes secretion from are well-known in the art and may be used mellitus. An additional highly preferred pancreatic beta cells. or routinely modified to assess the ability of indication is a complication associated with polypeptides of the invention (including diabetes (e.g., diabetic retinopathy, diabetic antibodies and agonists or antagonists of the nephropathy, kidney disease (e.g., renal failure, invention) to stimulate insulin secretion. nephropathy and/or other diseases and disorders For example, insulin secretion is measured as described in the ““Renal Disorders”” section by FMAT using anti-rat insulin antibodies. below), diabetic neuropathy, nerve disease and Insulin secretion from pancreatic beta cells nerve damage (e.g., due to diabetic neuropathy), is upregulated by glucose and also by blood vessel blockage, heart disease, stroke, certain proteins/peptides, and disregulation impotence (e.g., due to diabetic neuropathy or is a key component in diabetes. Exemplary blood vessel blockage), seizures, mental assays that may be used or routinely confusion, drowsiness, nonketotic modified to test for stimulation of insulin hyperglycemic-hyperosmolar coma, secretion (from pancreatic cells) by cardiovascular disease (e.g., heart disease, polypeptides of the invention (including atherosclerosis, microvascular disease, antibodies and agonists or antagonists of the hypertension, stroke, and other diseases and invention) include assays disclosed in: disorders as described in the ““Cardiovascular Ahren, B., et al., Am J Physiol, 277(4 Pt Disorders”” section below), dyslipidemia, 2): R959-66 (1999); Li, M., et al., endocrine disorders (as described in the Endocrinology, 138(9): 3735-40 (1997); ““Endocrine Disorders”” section below), Kim, K. H., et al., FEBS Lett, 377(2): 237-9 neuropathy, vision impairment (e.g., diabetic (1995); and, Miraglia S et. al., Journal of retinopathy and blindness), ulcers and impaired Biomolecular Screening, 4: 193-204 (1999), wound healing, and infection (e.g., infectious the contents of each of which is herein diseases and disorders as described in the incorporated by reference in its entirety. ““Infectious Diseases”” section below, Pancreatic cells that may be used according especially of the urinary tract and skin), carpal to these assays are publicly available (e.g., tunnel syndrome and Dupuytren's contracture). through the ATCC ™) and/or may be An additional highly preferred indication is routinely generated. Exemplary pancreatic obesity and/or complications associated with cells that may be used according to these obesity. Additional highly preferred indications assays include rat INS-1 cells. INS-1 cells include weight loss or alternatively, weight are a semi-adherent cell line established gain. Aditional highly preferred from cells isolated from an X-ray induced indications are complications associated with rat transplantable insulinoma. These cells insulin resistance. retain characteristics typical of native pancreatic beta cells including glucose inducible insulin secretion. References: Asfari et al. Endocrinology 1992 130: 167. 142 TNFa in Human T- cell 293T 143 TNFa in Human T- cell 2B9 144 Upregulation of CD152 FMAT. CD152 (a.k.a. CTLA-4) A highly preferred embodiment of the invention CD152 and activation expression is restricted to activated T cells. includes a method for activating T cells. An of T cells CD152 is a negative regulator of T cell alternative highly preferred embodiment of the proliferation. Reduced CD152 expression invention includes a method for inhibiting the has been linked to hyperproliferative and activation of and/or inactivating T cells. A autoimmune diseases. Overexpression of highly preferred embodiment of the invention CD152 may lead to impaired includes a method for inhibiting T cell immunoresponses. Assays for proliferation. An alternative highly preferred immunomodulatory proteins important in embodiment of the invention includes a method the maintenance of T cell homeostasis and for stimulating T cell proliferation. Highly expressed almost exclusively on CD4+ and preferred indications include blood disorders CD8+ T cells are well known in the art and (e.g., as described below under “Immune may be used or routinely modified to assess Activity”, “Blood-Related Disorders”, and/or the ability of polypeptides of the invention ““Cardiovascular Disorders””), Highly (including antibodies and agonists or preferred indications include autoimmune antagonists of the invention) to modulate diseases (e.g., rheumatoid arthritis, systemic the activation of T cells, maintain T cell lupus erythematosis, multiple sclerosis and/or homeostasis, and/or mediate humoral or as described below), immunodeficiencies (e.g., cell-mediated immunity. Exemplary assays as described below), boosting a T cell-mediated that test for immunomodulatory proteins immune response, and suppressing a T cell- evaluate the upregulation of cell surface mediated immune response. Highly markers, such as CD152, and the activation preferred indications include neoplastic diseases of T cells. Such assays that may be used or (e.g., leukemia, lymphoma, and/or as described routinely modified to test below under “Hyperproliferative Disorders”). immunomodulatory activity of polypeptides Additionally, highly preferred indications of the invention (including antibodies and include neoplasms and cancers, such as, for agonists or antagonists of the invention) example, leukemia, lymphoma, melanoma, and include, for example, the assays disclosed in prostate, breast, lung, colon, pancreatic, Miraglia et al., J Biomolecular Screening esophageal, stomach, brain, liver and urinary 4: 193-204 (1999); Rowland et al., cancer. Other preferred indications include ““Lymphocytes: a practical approach”” benign dysproliferative disorders and pre- Chapter 6: 138-160 (2000); McCoy et al., neoplastic conditions, such as, for example, Immunol Cell Biol 77(1): 1-10 (1999); hyperplasia, metaplasia, and/or dysplasia. Oostervegal et al., Curr Opin Immunol Preferred indications include anemia, 11(3): 294-300 (1999); and Saito T, Curr pancytopenia, leukopenia, thrombocytopenia, Opin Immunol 10(3): 313-321 (1998), the Hodgkin's disease, acute lymphocytic anemia contents of each of which are herein (ALL), plasmacytomas, multiple myeloma, incorporated by reference in its entirety. Burkitt's lymphoma, arthritis, AIDS, Human T cells that may be used according granulomatous disease, inflammatory bowel to these assays may be isolated using disease, sepsis, neutropenia, neutrophilia, techniques disclosed herein or otherwise psoriasis, immune reactions to transplanted known in the art. Human T cells are organs and tissues, hemophilia, primary human lymphocytes that mature in hypercoagulation, diabetes mellitus, the thymus and express a T Cell receptor endocarditis, meningitis, Lyme Disease, and CD3, CD4, or CD8. These cells inflammation and inflammatory disorders, and mediate humoral or cell-mediated immunity asthma and allergy. An additional preferred and may be preactivated to enhance indication is infection (e.g., as described below responsiveness to immunomodulatory under “Infectious Disease”). factors. 145 Upregulation of CD154 FMAT. CD154 (a.k.a., CD40L) CD154 and activation expression is induced following activation of T cells of T cells. Interaction between CD154 and CD40 on B cells is required for correct antibody class switching and germinal center formation. Mutations in CD154 are linked to immunodeficiencies and increased susceptibility to infections. Assays for immunomodulatory proteins important for antibody class switching and TH1 function and expressed on activated T helper lymphocytes are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, modulate antibody class switching, mediate TH1 function, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of cell surface markers, such as CD154, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Mackey et al., J Leukoc Biol 63(4): 418: 428 (1998); and Skov et al., 164(7): 3500-3505 (2000), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 146 Upregulation of CD69 FMAT. CD69 is an activation A highly preferred embodiment of the invention CD69 and activation marker that is expressed on activated T includes a method for activating T cells. An of T cells cells, B cells, and NK cells. CD69 is not alternative highly preferred embodiment of the expressed on resting T cells, B cells, or NK invention includes a method for inhibiting the cells. CD69 has been found to be associated activation of and/or inactivating T cells. A with inflammation. Assays for highly preferred embodiment of the invention immunomodulatory proteins expressed in T includes a method for activation B cells. An cells, B cells, and leukocytes are well alternative highly preferred embodiment of the known in the art and may be used or invention includes a method for inhibiting the routinely modified to assess the ability of activation of and/or inactivating B cells. A polypeptides of the invention (including highly preferred embodiment of the invention antibodies and agonists or antagonists of the includes a method for activating NK cells. An invention) to modulate the activation of T alternative highly preferred embodiment of the cells, and/or mediate humoral or cell- invention includes a method for inhibiting mediated immunity. Exemplary assays that activation of and/or inactivation NK cells. test for immunomodulatory proteins Highly preferred indications include evaluate the upregulation of cell surface inflammation and inflammatory disorders (e.g., markers, such as CD69, and the activation as described below under “Immune Activity”). of T cells. Such assays that may be used or Preferred indications include blood disorders routinely modified to test (e.g., as described below under “Immune immunomodulatory activity of polypeptides Activity”, “Blood-Related Disorders”, and/or of the invention (including antibodies and ““Cardiovascular Disorders””). Highly preferred agonists or antagonists of the invention) indications include autoimmune diseases (e.g., include, for example, the assays disclosed in rheumatoid arthritis, systemic lupus Miraglia et al., J Biomolecular Screening erythematosis, multiple sclerosis and/or as 4: 193-204 (1999); Rowland et al., described below), immunodeficiencies (e.g., as ““Lymphocytes: a practical approach”” described below), boosting a T cell-mediated Chapter 6: 138-160 (2000); Ferenczi et al., J immune response and alternatively suppressing Autoimmun 14(1): 63-78 (200); Werfel et a T cell-mediated immune response, and al., Allergy 52(4): 465-469 (1997); Taylor- boosting a B cell-mediated immune response Fishwick and Siegel, Eur J Immunol and alternatively suppressing a B cell-mediated 25(12): 3215-3221 (1995); and Afetra et al., immune response. An additional highly Ann Rheum Dis 52(6): 457-460 (1993), the preferred indication includes infection (e.g., as contents of each of which are herein described below under “Infectious Disease”). incorporated by reference in its entirety. Preferred indications also include anemia, Human T cells that may be used according pancytopenia, leukopenia, thrombocytopenia, to these assays may be isolated using Hodgkin's disease, acute lymphocytic anemia techniques disclosed herein or otherwise (ALL), plasmacytomas, multiple myeloma, known in the art. Human T cells are Burkitt's lymphoma, arthritis, AIDS, primary human lymphocytes that mature in granulomatous disease, inflammatory bowel the thymus and express a T Cell receptor disease, sepsis, neutropenia, neutrophilia, and CD3, CD4, or CD8. These cells psoriasis, suppression of immune reactions to mediate humoral or cell-mediated immunity transplanted organs and tissues, hemophilia, and may be preactivated to enhance hypercoagulation, diabetes mellitus, responsiveness to immunomodulatory endocarditis, meningitis, Lyme Disease, factors. inflammation and inflammatory disorders, asthma, and allergies. Preferred indications also include neoplastic diseases (e.g., leukemia, lymphoma, and/or as described below under “Hyperproliferative Disorders”). Preferred indications include neoplasms, such as, for example, leukemia, lymphoma, and prostate, breast, lung, colon, pancreatic, esophageal, stomach, brain, liver and urinary cancer. Other preferred indications include benign dysproliferative disorders and pre-neoplastic conditions, such as, for example, hyperplasia, metaplasia, and/or dysplasia. 147 Upregulation of CD71 FMAT. CD71 is the transferrin CD71 and activation receptor. Transferrin is a major iron of T cells carrying protein that is essential for cell proliferation. CD71 is expressed predominantly on cells that are actively proliferating. Assays for immunomodulatory proteins expressed on activated T cells, B cells, and most proliferating cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of cell surface markers, such as CD71, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); and Afetra et al., Ann Rheum Dis 52(6): 457-460 (1993), the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 148 Upregulation of HLA-DR FMAT. MHC class II is essential HLA-DR and for correct presentation of antigen to CD4+ activation of T cells T cells. Deregulation of MHC class II has been associated with autoimmune diseases (e.g., diabetes, rheumatoid arthritis, systemic lupus erythematosis, and multiple sclerosis). Assays for immunomodulatory proteins expressed on MHC class II expressing T cells and antigen presenting cells are well known in the art and may be used or routinely modified to assess the ability of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) to modulate the activation of T cells, and/or mediate humoral or cell-mediated immunity. Exemplary assays that test for immunomodulatory proteins evaluate the upregulation of MHC class II products, such as HLA-DR antigens, and the activation of T cells. Such assays that may be used or routinely modified to test immunomodulatory activity of polypeptides of the invention (including antibodies and agonists or antagonists of the invention) include, for example, the assays disclosed in Miraglia et al., J Biomolecular Screening 4: 193-204 (1999); Rowland et al., “Lymphocytes: a practical approach” Chapter 6: 138-160 (2000); Lamour et al., Clin Exp Immunol 89(2): 217-222 (1992); Hurme and Sihvola, Immunol Lett 20(3): 217-222 (1989); Gansbacher and Zier, Cell Immunol 117(1): 22-34 (1988); and Itoh et al., J Histochem Cytochem 40(11): 1675-1683, the contents of each of which are herein incorporated by reference in its entirety. Human T cells that may be used according to these assays may be isolated using techniques disclosed herein or otherwise known in the art. Human T cells are primary human lymphocytes that mature in the thymus and express a T Cell receptor and CD3, CD4, or CD8. These cells mediate humoral or cell-mediated immunity and may be preactivated to enhance responsiveness to immunomodulatory factors. 149 VEGF in SW480

Table 1F: Polynucleotides encoding polypeptides of the present invention can be used in assays to test for one or more biological activities. One such biological activity which may be tested includes the ability of polynucleotides and polypeptides of the invention to stimulate up-regulation or down-regulation of expression of particular genes and proteins. Hence, if polynucleotides and polypeptides of the present invention exhibit activity in altering particular gene and protein expression patterns, it is likely that these polynucleotides and polypeptides of the present invention may be involved in, or capable of effecting changes in, diseases associated with the altered gene and protein expression profiles. Hence, polynucleotides, polypeptides, or antibodies of the present invention could be used to treat said associated diseases.

TaqMan® assays may be performed to assess the ability of polynucleotides (and polypeptides they encode) to alter the expression pattern of particular “target” genes. TaqMan® reactions are performed to evaluate the ability of a test agent to induce or repress expression of specific genes in different cell types. TaqMan® gene expression quantification assays (“TaqMan® assays”) are well known to, and routinely performed by, those of ordinary skill in the art. TaqMan® assays are performed in a two step reverse transcription/polymerase chain reaction (RT-PCR). In the first (RT) step, cDNA is reverse transcribed from total RNA samples using random hexamer primers. In the second (PCR) step, PCR products are synthesized from the cDNA using gene specific primers.

To quantify gene expression the TaqMan® PCR reaction exploits the 5′ nuclease activity of AmpliTaq Gold® DNA Polymerase to cleave a TaqMan® probe (distinct from the primers) during PCR. The TaqMan® probe contains a reporter dye at the 5′-end of the probe and a quencher dye at the 3′ end of the probe. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. AmpliTaq Gold® DNA Polymerase then cleaves the probe between the reporter and quencher when the probe hybridizes to the target, resulting in increased fluorescence of the reporter (see FIG. 2). Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye.

After the probe fragments are displaced from the target, polymerization of the strand continues. The 3′-end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. The increase in fluorescence signal is detected only if the target sequence is complementary to the probe and is amplified during PCR. Because of these requirements, any nonspecific amplification is not detected.

For test sample preparation, vector controls or constructs containing the coding sequence for the gene of interest are transfected into cells, such as for example 293T cells, and supernatants collected after 48 hours. For cell treatment and RNA isolation, multiple primary human cells or human cell lines are used; such cells may include but are not limited to, Normal Human Dermal Fibroblasts, Aortic Smooth Muscle, Human Umbilical Vein Endothelial Cells, HepG2, Daudi, Jurkat, U937, Caco, and THP-1 cell lines. Cells are plated in growth media and growth is arrested by culturing without media change for 3 days, or by switching cells to low serum media and incubating overnight. Cells are treated for 1, 6, or 24 hours with either vector control supernatant or sample supernatant (or purified/partially purified protein preparations in buffer). Total RNA is isolated; for example, by using TRIZOL™ extraction or by using the Ambion RNAqueous™-4PCR RNA isolation system. Expression levels of multiple genes are analyzed using TaqMan®, and expression in the test sample is compared to control vector samples to identify genes induced or repressed. Each of the above described techniques are well known to, and routinely performed by, those of ordinary skill in the art.

Table 1F indicates particular disease classes and preferred indications for which polynucleotides, polypeptides, or antibodies of the present invention may be used in detecting, diagnosing, preventing, treating and/or ameliorating said diseases and disorders based on “target” gene expression patterns which may be up- or down-regulated by polynucleotides (and the encoded polypeptides) corresponding to each indicated cDNA Clone ID (shown in Table 1F, Column 2).

Thus, in preferred embodiments, the present invention encompasses a method of detecting, diagnosing, preventing, treating, and/or ameliorating a disease or disorder listed in the “Disease Class” and/or “Preferred Indication” columns of Table 1F; comprising administering to a patient in which such detection, diagnosis, prevention, or treatment is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to detect, diagnose, prevent, treat, or ameliorate the disease or disorder. The first and second columns of Table 1F shows the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in detecting, diagnosing, preventing, treating, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in the “Disease Class” or “Preferred Indication” Columns of Table 1F.

In another embodiment, the present invention also encompasses methods of detecting, diagnosing, preventing, treating, or ameliorating a disease or disorder listed in the “Disease Class” or “Preferred Indication” Columns of Table 1F; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in the “Disease Class” or “Preferred Indication” Columns of Table 1F.

The “Disease Class” Column of Table 1F provides a categorized descriptive heading for diseases, disorders, and/or conditions (more fully described below) that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).

The “Preferred Indication” Column of Table 1F describes diseases, disorders, and/or conditions that may be detected, diagnosed, prevented, treated, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).

The “Cell Line” and “Exemplary Targets” Columns of Table 1F indicate particular cell lines and target genes, respectively, which may show altered gene expression patterns (i.e., up- or down-regulation of the indicated target gene) in TaqMan® assays, performed as described above, utilizing polynucleotides of the cDNA Clone ID shown in the corresponding row. Alteration of expression patterns of the indicated “Exemplary Target” genes is correlated with a particular “Disease Class” and/or “Preferred Indication” as shown in the corresponding row under the respective column headings.

The “Exemplary Accessions” Column indicates GenBank Accessions (available online through the National Center for Biotechnology Information (NCBI) at the world wide web at ncbi.nlm.nih.gov/) which correspond to the “Exemplary Targets” shown in the adjacent row.

The recitation of “Cancer” in the “Disease Class” Column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate neoplastic diseases and/or disorders (e.g., leukemias, cancers, etc., as described below under “Hyperproliferative Disorders”).

The recitation of “Immune” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, prevent, treat, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).

The recitation of “Angiogenesis” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), diseases and/or disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”), diseases and/or disorders involving cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), diseases and/or disorders involving angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), to promote or inhibit cell or tissue regeneration (e.g., as described below under “Regeneration”), or to promote wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”). Highly preferred indications include diagnosis, prevention, treatment, and/or amelioration of diseases and disorders involving angiogenesis, wound healing, neoplasia (particularly including, but not limited to, tumor metastases), and cardiovascular diseases and disorders; as described herein under the headings “Hyperproliferative Disorders,” “Regeneration,” “Anti-Angiogenesis Activity,” “Diseases at the Cellular Level,” and “Wound Healing and Epithelial Cell Proliferation.”

The recitation of “Diabetes” in the “Disease Class” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to detect, diagnose, treat, prevent, and/or ameliorate diabetes (including diabetes mellitus types I and II), as well as diseases and/or disorders associated with, or consequential to, diabetes (e.g. as described below under “Endocrine Disorders,” “Renal Disorders,” and “Gastrointestinal Disorders”).

TABLE 1F Gene cDNA Exemplary No. Clone ID Disease Class Preferred Indications Cell Line Targets Exemplary Accessions 14 HJACG02 Angiogenesis Highly preferred Adipocytes- ICAM gb|X06990|HSICAM1 indications include 3/12/01 PAI gb|X12701|HSENDPAI diagnosis, prevention, Vegf1 gb|AF024710|AF024710 treatment, and/or amelioration of diseases and disorders involving angiogenesis, wound healing, neoplasia (particularly including, but not limited to, tumor metastases), and cardiovascular diseases and disorders; as described herein under the headings “Hyperproliferative Disorders,” “Regeneration,” “Anti- Angiogenesis Activity,” “Diseases at the Cellular Level,” and “Wound Healing and Epithelial Cell Proliferation.” 14 HJACG02 Angiogenesis Highly preferred AOSMC VCAM gb|A30922|A30922 indications include diagnosis, prevention, treatment, and/or amelioration of diseases and disorders involving angiogenesis, wound healing, neoplasia (particularly including, but not limited to, tumor metastases), and cardiovascular diseases and disorders; as described herein under the headings “Hyperproliferative Disorders,” “Regeneration,” “Anti- Angiogenesis Activity,” “Diseases at the Cellular Level,” and “Wound Healing and Epithelial Cell Proliferation.”(AOSMC cells are aortic smooth muscle cells). 14 HJACG02 Angiogenesis Highly preferred Daudi ICAM gb|X06990|HSICAM1 indications include VCAM gb|A30922|A30922 diagnosis, prevention, treatment, and/or amelioration of diseases and disorders involving angiogenesis, wound healing, neoplasia (particularly including, but not limited to, tumor metastases), and cardiovascular diseases and disorders; as described herein under the headings “Hyperproliferative Disorders,” “Regeneration,” “Anti- Angiogenesis Activity,” “Diseases at the Cellular Level,” and “Wound Healing and Epithelial Cell Proliferation.”(The Daudi cell line is a human B lymphoblast cell line available through the ATCC ™ as cell line number CCL-213). 14 HJACG02 Angiogenesis Highly preferred HUVEC ICAM gb|X06990|HSICAM1 indications include TSP-1 gb|X04665|HSTHROMR diagnosis, prevention, Vegf1 gb|AF024710|AF024710 treatment, and/or amelioration of diseases and disorders involving angiogenesis, wound healing, neoplasia (particularly including, but not limited to, tumor metastases), and cardiovascular diseases and disorders; as described herein under the headings “Hyperproliferative Disorders,” “Regeneration,” “Anti- Angiogenesis Activity,” “Diseases at the Cellular Level,” and “Wound Healing and Epithelial Cell Proliferation.”(HUVEC cells are human umbilical vein endothelial cells). 14 HJACG02 Cancer Highly preferred Adipocytes- Egr1 indications include 3/12/01 neoplastic diseases (e.g. cancer) such as described herein under the heading “Hyperproliferative Disordersî (particularly including, but not limited to, cancer involving adipocytes). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating cancer and hyperproliferative disorders. (Primary adipocytes) 14 HJACG02 Cancer Highly preferred AOSMC M1 RIBO R gb|X59543|HSRIREM1 indications include neoplastic diseases (e.g. cancer) such as described herein under the heading “Hyperproliferative Disordersî (particularly including, but not limited to, cancers of muscle tissues and the cardiovascular system (e.g. heart, lungs, circulatory system)). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating cancer and hyperproliferative disorders. (AOSMC cells are aortic smooth muscle cells). 14 HJACG02 Cancer Highly preferred Daudi Cyclin A1 gb|U97680|HSU97680 indications include neoplastic diseases (e.g. cancer) such as described herein under the heading “Hyperproliferative Disordersî (particularly including, but not limited to, cancers of immune cells, such as B-cells). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating cancer and hyperproliferative disorders involving immune cells (such as B- cells). (The Daudi cell line is a human B lymphoblast cell line available through the ATCC ™ as cell line number CCL-213). 14 HJACG02 Cancer Highly preferred HEK293 E-cadherin gb|Z35408|HSECAD9 indications include neoplastic diseases (e.g. cancer) such as described herein under the heading “Hyperproliferative Disordersî (particularly including, but not limited to, cancers of epithelial cells or cancers involving the renal system). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating cancer and hyperproliferative disorders involving epithelial cells or the renal system. (The 293 cell line human embryonal kidney epithelial cell line available through the ATCC ™ as cell line number CRL-1573). 14 HJACG02 Cancer Highly preferred Jurkat Cyclin A1 gb|U97680|HSU97680 indications include neoplastic diseases (e.g. cancer) such as described herein under the heading “Hyperproliferative Disordersî (particularly including, but not limited to, cancers of immune cells, such as T-cells). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating cancer and hyperproliferative disorders involving immune cells (such as T- cells). (The Jurkat cell line is a human T lymphocyte cell line available through the ATCC ™ as cell line number TIB-152). 14 HJACG02 Cancer Highly preferred Liver Cyclin D2 gb|X68452|HSCYCD2 indications include neoplastic diseases (e.g. cancer) such as described herein under the heading “Hyperproliferative Disordersî (particularly including, but not limited to, cancers involving cells of the hepatic system). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating cancer and hyperproliferative disorders involving cells of the hepatic system. 14 HJACG02 Cancer Highly preferred NHDF Cyclin A1 gb|U97680|HSU97680 indications include neoplastic diseases (e.g. cancer) such as described herein under the heading “Hyperproliferative Disordersî (particularly including, but not limited to cancers involving cells of the skin). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating cancer and hyperproliferative disorders involving skin cells. (NHDF cells are normal human dermal fibroblasts). 14 HJACG02 Diabetes A highly preferred Adipocytes CAP gb|AF136380|AF136380 indication is diabetes. (4D)- PEPCK1 gb|L05144|HUMPHOCAR Additional highly 09/01/01 preferred indications include complications associated with diabetes (e.g., diabetic retinopathy, diabetic nephropathy, kidney disease (e.g., renal failure, nephropathy and/or other diseases and disorders as described in the “Renal Disorders” section below), diabetic neuropathy, nerve disease and nerve damage (e.g., due to diabetic neuropathy), blood vessel blockage, heart disease, stroke, impotence (e.g., due to diabetic neuropathy or blood vessel blockage), seizures, mental confusion, drowsiness, nonketotic hyperglycemic- hyperosmolar coma, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the “Cardiovascular Disorders” section below), dyslipidemia, endocrine disorders (as described in the “Endocrine Disorders” section below), neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, and infection (e.g., infectious diseases and disorders as described in the “Infectious Diseases” section below, especially of the urinary tract and skin). Highly preferred indications also include obesity, weight gain, and weight loss, as well as complications associated with obesity, weight gain, and weight loss. Preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating the above mentioned conditions, disorders, and diseases. 14 HJACG02 Diabetes A highly preferred Adipocytes- CAP gb|AF136380|AF136380 indication is diabetes. 3/12/01 Hexokinase gb|Z46354|HSHKEX1 Additional highly II preferred indications include complications associated with diabetes (e.g., diabetic retinopathy, diabetic nephropathy, kidney disease (e.g., renal failure, nephropathy and/or other diseases and disorders as described in the “Renal Disorders” section below), diabetic neuropathy, nerve disease and nerve damage (e.g., due to diabetic neuropathy), blood vessel blockage, heart disease, stroke, impotence (e.g., due to diabetic neuropathy or blood vessel blockage), seizures, mental confusion, drowsiness, nonketotic hyperglycemic- hyperosmolar coma, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the “Cardiovascular Disorders” section below), dyslipidemia, endocrine disorders (as described in the “Endocrine Disorders” section below), neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, and infection (e.g., infectious diseases and disorders as described in the “Infectious Diseases” section below, especially of the urinary tract and skin). Highly preferred indications also include obesity, weight gain, and weight loss, as well as complications associated with obesity, weight gain, and weight loss. Preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating the above mentioned conditions, disorders, and diseases. 14 HJACG02 Diabetes A highly preferred AOSMC IRS1 gb|X90563|HSPPARGAM indication is diabetes. PPARg Additional highly preferred indications include complications associated with diabetes (e.g., diabetic retinopathy, diabetic nephropathy, kidney disease (e.g., renal failure, nephropathy and/or other diseases and disorders as described in the “Renal Disorders” section below), diabetic neuropathy, nerve disease and nerve damage (e.g., due to diabetic neuropathy), blood vessel blockage, heart disease, stroke, impotence (e.g., due to diabetic neuropathy or blood vessel blockage), seizures, mental confusion, drowsiness, nonketotic hyperglycemic- hyperosmolar coma, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the “Cardiovascular Disorders” section below), dyslipidemia, endocrine disorders (as described in the “Endocrine Disorders” section below), neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, and infection (e.g., infectious diseases and disorders as described in the “Infectious Diseases” section below, especially of the urinary tract and skin). Highly preferred indications also include obesity, weight gain, and weight loss, as well as complications associated with obesity, weight gain, and weight loss. Preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating the above mentioned conditions, disorders, and diseases. (AOSMC cells are human aortic smooth muscle cells). 14 HJACG02 Diabetes A highly preferred Liver Glucose6 gb|U91844|CFU91844 indication is diabetes. phosphatase Additional highly preferred indications include complications associated with diabetes (e.g., diabetic retinopathy, diabetic nephropathy, kidney disease (e.g., renal failure, nephropathy and/or other diseases and disorders as described in the “Renal Disorders” section below), diabetic neuropathy, nerve disease and nerve damage (e.g., due to diabetic neuropathy), blood vessel blockage, heart disease, stroke, impotence (e.g., due to diabetic neuropathy or blood vessel blockage), seizures, mental confusion, drowsiness, nonketotic hyperglycemic- hyperosmolar coma, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the “Cardiovascular Disorders” section below), dyslipidemia, endocrine disorders (as described in the “Endocrine Disorders” section below), neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, and infection (e.g., infectious diseases and disorders as described in the “Infectious Diseases” section below, especially of the urinary tract and skin). Highly preferred indications also include obesity, weight gain, and weight loss, as well as complications associated with obesity, weight gain, and weight loss. Preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating the above mentioned conditions, disorders, and diseases. 14 HJACG02 Immune Highly preferred Adipocytes- ICAM gb|X06990|HSICAM1 indications include 3/12/01 Il6 gb|X04403|HS26KDAR immunological disorders Rag1 gb|M29474|HUMRAG1 such as described herein under the heading “Immune Activity” and/or “Blood-Related Disorders” (particularly including, but not limited to, immune disorders involving adipocytes). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating disorders of the immune system (particularly including, but not limited to, immune disorders involving adipocytes). 14 HJACG02 Immune Highly preferred AOSMC CD30 gb|AJ300189|HSA30018 indications include CD40 gb|X02532|HSIL1BR immunological disorders IL1B gb|X12705|HSBCDFIA such as described herein IL5 gb|AJ270944|HSA27094 under the heading TNF gb|A30922|A30922 “Immune Activity” and/or VCAM “Blood-Related Disorders” (particularly including, but not limited to, immune disorders involving muscle tissues and the cardiovascular system (e.g. heart, lungs, circulatory system)). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating disorders of the immune system (particularly including, but not limited to, immune disorders involving muscle tissue or the cardiovascular system). (AOSMC cells are human aortic smooth muscle cells). 14 HJACG02 Immune Highly preferred Caco-2 Rag1 gb|M29474|HUMRAG1 indications include immunological disorders such as described herein under the heading “Immune Activity” and/or “Blood-Related Disorders” (particularly including, but not limited to, immune disorders involving the cells of the gastrointestinal tract). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating disorders of the immune system (particularly including, but not limited to, immune disorders involving cells of the gastrointestinal tract). (The Caco-2 cell line is a human colorectal adenocarcinoma cell line available through the ATCC ™ as cell line number HTB-37). 14 HJACG02 Immune Highly preferred Daudi ICAM gb|X06990|HSICAM1 indications include Rag1 gb|M29474|HUMRAG1 immunological disorders VCAM gb|A30922|A30922 such as described herein under the heading “Immune Activity” and/or “Blood-Related Disorders” (particularly including, but not limited to, immune disorders involving the B-cells). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating disorders of the immune system (particularly including, but not limited to, immune disorders involving B-cells). (The Daudi cell line is a human B lymphoblast cell line available through the ATCC ™ as cell line number CCL-213). 14 HJACG02 Immune Highly preferred HEK293 c-maf gb|AF055377|AF055377 indications include immunological disorders such as described herein under the heading “Immune Activity” and/or “Blood-Related Disorders” (particularly including, but not limited to, immune disorders involving epithelial cells or the renal system). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating disorders of the immune system (particularly including, but not limited to, immune disorders involving epithelial cells or the renal system). (The 293 cell line is a human embryonal kidney epithelial cell line available through the ATCC ™ as cell line number CRL-1573). 14 HJACG02 Immune Highly preferred HUVEC ICAM gb|X06990|HSICAM1 indications include immunological disorders such as described herein under the heading “Immune Activity” and/or “Blood-Related Disorders” (particularly including, but not limited to, immune disorders involving endothelial cells). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating disorders of the immune system (particularly including, but not limited to, immune disorders involving endothelial cells). (HUVEC cells are human umbilical vein endothelial cells). 14 HJACG02 Immune Highly preferred Jurkat Rag2 gb|AY011962|AY011962 indications include TNF gb|AJ270944|HSA27094 immunological disorders such as described herein under the heading “Immune Activity” and/or “Blood-Related Disorders” (particularly including, but not limited to, immune disorders involving T-cells). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating disorders of the immune system (particularly including, but not limited to, immune disorders involving T-cells). (The Jurkat cell line is a human T lymphocyte cell line available through the ATCC ™ as cell line number TIB-152). 14 HJACG02 Immune Highly preferred NHDF Rag1 gb|M29474|HUMRAG1 indications include immunological disorders such as described herein under the heading “Immune Activity” and/or “Blood-Related Disorders” (particularly including, but not limited to, immune disorders involving the skin). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating disorders of the immune system (particularly including, but not limited to, immune disorders involving the skin). (NHDF cells are normal human dermal fibroblasts). 14 HJACG02 Immune Highly preferred U937 GATA1 gb|X17254|HSERYF1 indications include IL5 gb|X12705|HSBCDFIA immunological disorders TNF gb|AJ270944|HSA27094 such as described herein under the heading “Immune Activity” and/or “Blood-Related Disorders” (particularly including, but not limited to, immune disorders involving monocytes). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating disorders of the immune system (particularly including, but not limited to, immune disorders involving monocytes). (The U937 cell line is a human monocyte cell line available through the ATCC ™ as cell line number CRL-1593.2). 14 HJACG02 Immune Highly preferred Adipocytes- ICAM gb|X06990|HSICAM1 indications include 3/12/01 I16 gb|X04403|HS26KDAR immunological disorders Rag1 gb|M29474|HUMRAG1 such as described herein under the heading “Immune Activity” and/or “Blood-Related Disorders” (particularly including, but not limited to, immune disorders involving adipocytes). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating disorders of the immune system (particularly including, but not limited to, immune disorders involving adipocytes). 14 HJACG02 Immune Highly preferred AOSMC CD30 indications include CD40 gb|AJ300189|HSA30018 immunological disorders IL1B gb|X02532|HSIL1BR such as described herein IL5 gb|X12705|HSBCDFIA under the heading TNF gb|AJ270944|HSA27094 “Immune Activity” and/or VCAM gb|A30922|A30922 “Blood-Related Disorders” (particularly including, but not limited to, immune disorders involving muscle tissues and the cardiovascular system (e.g. heart, lungs, circulatory system)). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating disorders of the immune system (particularly including, but not limited to, immune disorders involving muscle tissue or the cardiovascular system). (AOSMC cells are human aortic smooth muscle cells). 14 HJACG02 Immune Highly preferred Caco-2 Rag1 gb|M29474|HUMRAG1 indications include immunological disorders such as described herein under the heading “Immune Activity” and/or “Blood-Related Disorders” (particularly including, but not limited to, immune disorders involving the cells of the gastrointestinal tract). Highly preferred embodiments of the invention include methods of preventing, detecting, diagnosing, treating and/or ameliorating disorders of the immune system (particularly including, but not limited to, immune disorders involving cells of the gastrointestinal tract). (The Caco-2 cell line is a human colorectal adenocarcinoma cell line available through the ATCC ™ as cell line number HTB-37).

Table 2 further characterizes certain encoded polypeptides of the invention, by providing the results of comparisons to protein and protein family databases. The first column provides a unique clone identifier, “Clone ID NO:”, corresponding to a cDNA clone disclosed in Tables 1A, 1B.1, and/or 1B.2. The second column provides the unique contig identifier, “Contig ID:” which allows correlation with the information in Tables 1B.1 and 1B.2. The third column provides the sequence identifier, “SEQ ID NO:”, for the contig polynucleotide sequences. The fourth column provides the analysis method by which the homology/identity disclosed in the Table was determined. The fifth column provides a description of the PFAM/NR hit identified by each analysis. Column six provides the accession number of the PFAM/NR hit disclosed in the fifth column. Column seven, score/percent identity, provides a quality score or the percent identity, of the hit disclosed in column five. Comparisons were made between polypeptides encoded by polynucleotides of the invention and a non-redundant protein database (herein referred to as “NR”), or a database of protein families (herein referred to as “PFAM”), as described below.

The NR database, which comprises the NBRF PIR database, the NCBI GenPept database, and the SIB SwissProt and TrEMBL databases, was made non-redundant using the computer program nrdb2 (Warren Gish, Washington University in Saint Louis). Each of the polynucleotides shown in Tables 1B.1 and 1B.2, column 3 (e.g., SEQ ID NO:X or the ‘Query’ sequence) was used to search against the NR database. The computer program BLASTX was used to compare a 6-frame translation of the Query sequence to the NR database (for information about the BLASTX algorithm please see Altshul et al., J. Mol. Biol. 215:403-410 (1990), and Gish and States, Nat. Genet. 3:266-272 (1993). A description of the sequence that is most similar to the Query sequence (the highest scoring ‘Subject’) is shown in column five of Table 2 and the database accession number for that sequence is provided in column six. The highest scoring ‘Subject’ is reported in Table 2 if (a) the estimated probability that the match occurred by chance alone is less than 1.0e-07, and (b) the match was not to a known repetitive element. BLASTX returns alignments of short polypeptide segments of the Query and Subject sequences which share a high degree of similarity; these segments are known as High-Scoring Segment Pairs or HSPs. Table 2 reports the degree of similarity between the Query and the Subject for each HSP as a percent identity in Column 7. The percent identity is determined by dividing the number of exact matches between the two aligned sequences in the HSP, dividing by the number of Query amino acids in the HSP and multiplying by 100. The polynucleotides of SEQ ID NO:X which encode the polypeptide sequence that generates an HSP are delineated by columns 8 and 9 of Table 2.

The PFAM database, PFAM version 2.1, (Sonnhammer, Nucl. Acids Res., 26:320-322, 1998)) consists of a series of multiple sequence alignments; one alignment for each protein family. Each multiple sequence alignment is converted into a probability model called a Hidden Markov Model, or HMM, that represents the position-specific variation among the sequences that make up the multiple sequence alignment (see, e.g., Durbin, et al., Biological sequence analysis: probabilistic models of proteins and nucleic acids, Cambridge University Press, 1998 for the theory of HMMs). The program HMMER version 1.8 (Sean Eddy, Washington University in Saint Louis) was used to compare the predicted protein sequence for each Query sequence (SEQ ID NO:Y in Table 1B.1) to each of the HMMs derived from PFAM version 2.1. A HMM derived from PFAM version 2.1 was said to be a significant match to a polypeptide of the invention if the score returned by HMMER 1.8 was greater than 0.8 times the HMMER 1.8 score obtained with the most distantly related known member of that protein family. The description of the PFAM family which shares a significant match with a polypeptide of the invention is listed in column 5 of Table 2, and the database accession number of the PFAM hit is provided in column 6. Column 7 provides the score returned by HMMER version 1.8 for the alignment. Columns 8 and 9 delineate the polynucleotides of SEQ ID NO:X which encode the polypeptide sequence which show a significant match to a PFAM protein family.

As mentioned, columns 8 and 9 in Table 2, “NT From” and “NT To”, delineate the polynucleotides of “SEQ ID NO:X” that encode a polypeptide having a significant match to the PFAM/NR database as disclosed in the fifth column. In one embodiment, the invention provides a protein comprising, or alternatively consisting of, a polypeptide encoded by the polynucleotides of SEQ ID NO:X delineated in columns 8 and 9 of Table 2. Also provided are polynucleotides encoding such proteins, and the complementary strand thereto.

The nucleotide sequence SEQ ID NO:X and the translated SEQ ID NO:Y are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, the nucleotide sequences of SEQ ID NO:X are useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:X or the cDNA contained in ATCC™ Deposit No:Z. These probes will also hybridize to nucleic acid molecules in biological samples, thereby enabling immediate applications in chromosome mapping, linkage analysis, tissue identification and/or typing, and a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from SEQ ID NO:Y may be used to generate antibodies which bind specifically to these polypeptides, or fragments thereof, and/or to the polypeptides encoded by the cDNA clones identified in, for example, Table 1A and/or 1B.

Nevertheless, DNA sequences generated by sequencing reactions can contain sequencing errors. The errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence. The erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence. In these cases, the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1000 bases).

Accordingly, for those applications requiring precision in the nucleotide sequence or the amino acid sequence, the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:X, and a predicted translated amino acid sequence identified as SEQ ID NO:Y, but also a sample of plasmid DNA containing cDNA ATCC™ Deposit No:Z (e.g., as set forth in columns 2 and 3 of Table 1A and/or as set forth, for example, in Tables 6 and 7). The nucleotide sequence of each deposited clone can readily be determined by sequencing the deposited clone in accordance with known methods. Further, techniques known in the art can be used to verify the nucleotide sequences of SEQ ID NO:X. The predicted amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by a particular clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human cDNA, collecting the protein, and determining its sequence.

TABLE 2 SEQ ID PFam/NR Score/ cDNA Contig NO: Analysis Accession Percent NT NT Clone ID ID: X Method PFam/NR Description Number Identify From To HTEEB42 206980 9 HMMER 2.1.1 PFAM: Immunoglobulin domain PF00047 48.5 500 706 HTEEB42 206980 9 WUblastx.64 (AAG49022) Junctional adhesion AAG49022 94% 110 952 molecule 2. HTEEB42 206980 9 WUblastx.64 (AAG49022) Junctional adhesion AAG49022 99% 59 952 molecule 2. HEMCM42 407085 11 WUblastx.64 (Q9QZW3) TYPE I Q9QZW3 91% 372 443 TRANSMEMBRANE PROTEIN FN14. 88% 133 360 HEQCC55 884824 13 WUblastx.64 (Q9NP84) TYPE I Q9NP84 64% 62 397 TRANSMENMBRANE PROTEIN PRECURSOR (TYPE I TRANSMEMBRAN HEMAE80 409495 20 WUblastx.64 (Q9NRR1) CYTOKINE-LIKE PROTEIN Q9NRR1 100% 12 419 C17. HEMAE80 1310948 22 WUblastx.64 (Q9NRR1) CYTOKINE-LIKE PROTEIN Q9NRR1 100% 136 468 C17. HRDFB85 411020 28 HMMER 2.1.1 PFAM: PMP-22/EMP/MP20/Claudin PF00822 150.2 282 −233 family HRDFB85 411020 28 WUblastx.64 (AAH00671) Claudin 4. AAH00671 100% 209 835 HDTAW95 412472 38 WUblastx.64 (Q96CG8) Similar to RIKEN cDNA Q96CG8 100% 130 249 1110014B07 gene. 97% 230 856 HEMCV19 423219 46 HMMER 2.1.1 PFAM: ATP1G1/PLM/MAT8 family PF02038 70.5 475 609 HEMCV19 423219 46 WUblastx.64 (Q96DB9) FXYD domain-containing ion FXY5_HUMAN 88% 79 612 transport regulator 5 p HETBX14 806447 61 HMMER 2.1.1 PFAM: Trypsin PF00089 281.2 270 935 HETBX14 806447 61 WUblastx.64 (Q9NS65) PROSTATE-TYPE Q9NS65 94% 111 956 HIPPOSTASIN. HETBX14 422659 63 WUblastx.64 (Q9NS65) PROSTATE-TYPE Q9NS65 89% 4 810 HIPPOSTASIN. HLHSK94 1307727 72 WUblastx.64 (AAH18037) Wnt inhibitory factor-1. AAH18037 90% 112 1248 HLHSK94 422828 74 HMMER 2.1.1 PFAM: EGF-like domain PF00008 43.9 −527 −610 HLHSK94 422828 74 WUblastx.64 (AAH18037) Wnt inhibitory factor-1. AAH18037 99% 112 1248 HLHFP03 460467 76 blastx.2 (AX058598) unnamed protein product emb|CAC22514.1| 95% 390 43 [Homo sapiens] HLHFP03 460467 78 WUblastx.64 (Q9WVC2) LY-6/NEUROTOXIN Q9WVC2 81% 224 571 HOMOLOG (ADULT MALE HIPPOCAMPUS CDNA, RIKEN HHTLF25 461438 83 WUblastx.64 (Q9UMT3) KILLER ACTIVATING Q9UMT3 91% 142 474 RECEPTOR ASSOCIATED PROTEIN, ISOFORM B. HHTLF25 461438 83 blastx.2 KILLER ACTIVATING RECEPTOR sp|Q9UMT3|Q9UMT3 99% 142 474 ASSOCIATED PROTEIN, ISOFORM B. HTADX17 457172 90 WUblastx.64 (AK009505) putative [Mus musculus] dbj|BAB26328.1| 67% 548 778 53% 490 585 58% 165 488 HTADX17 753289 92 WUblastx.64 (Q96A28) CD84-H1 (CD2 FAMILY 10). Q96A28 93% 92 412 79% 408 959 HTADX17 753289 92 WUblastx.64 (Q96A28) CD84-H1 (CD2 FAMILY 10). Q96A28 100% 92 412 99% 408 959 HTADX17 457172 94 WUblastx.64 (Q96A28) CD84-H1 (CD2 FAMILY 10). Q96A28 78% 490 585 97% 548 952 99% 84 488 HJACG02 509948 102 WUblastx.64 (Q9HD89) CYSTEINE-RICH Q9HD89 100% 47 370 SECRETED PROTEIN (C/EBP- EPSILON REGULATED MYEL HJACG02 509948 102 WUblastx.64 (Q9HD89) CYSTEINE-RICH Q9HD89 100% 92 370 SECRETED PROTEIN (C/EBP- EPSILON REGULATED MYEL HJACG02 1307789 104 WUblastx.64 (Q9HD89) CYSTEINE-RICH Q9HD89 100% 111 389 SECRETED PROTEIN (C/EBP- EPSILON REGULATED MYEL HJACG02 1307789 104 WUblastx.64 (Q9HD89) CYSTEINE-RICH Q9HD89 100% 66 389 SECRETED PROTEIN (C/EBP- EPSILON REGULATED MYEL HKGAJ54 498303 107 HMMER 2.1.1 PFAM: Immunoglobulin domain PF00047 52.8 481 657 HKGAJ54 498303 107 WUblastx.64 (AAL31867) Magic roundabout. AAL31867 96% 846 929 98% 31 849 HKGAJ54 498303 107 WUblastx.64 (BAB55411) CDNA FLJ14946 fis, clone BAB55411 96% 846 929 PLACE2000034, w 90% 109 849 HKGAJ54 1300770 109 WUblastx.64 (BAB55411) CDNA FLJ14946 fis, clone BAB55411 91% 102 923 PLACE2000034, w HSVAK93 597462 116 HMMER 2.1.1 PFAM: Immunoglobulin domain PF00047 23.6 117 362 HSVAK93 597462 116 WUblastx.64 (AAH08988) Unknown (protein for AAH08988 100% 21 530 MGC: 17333). HE8CH92 609866 118 HMMER 2.1.1 PFAM: Immunoglobulin domain PF00047 23.6 127 372 HE8CH92 609866 118 WUblastx.64 (AAH08988) Unknown (protein for AAH08988 100% 31 1164 MGC: 17333). HSDEK49 625998 123 HMMER 2.1.1 PFAM: Immunoglobulin domain PF00047 18.7 225 470 HSDEK49 625998 123 WUblastx.64 (Q9Y279) Z39IG PROTEIN Q9Y279 88% 444 1040 PRECURSOR. 99% 126 542 HSDEK49 1352253 125 WUblastx.64 (Q9Y279) Z39IG PROTEIN Q9Y279 100% 60 1256 PRECURSOR. HWBAO62 838164 127 HMMER 2.1.1 PFAM: Immunoglobulin domain PF00047 27.9 202 402 HWBAO62 838164 127 WUblastx.64 (Q14288) HYPOTHETICAL PROTEIN Q14288 45% 1331 1618 (FRAGMENT). 66% 1158 1334 62% 1847 1894 55% 1594 1839 HWBAO62 625914 129 WUblastx.64 (Q14288) HYPOTHETICAL PROTEIN Q14288 43% 1358 1645 (FRAGMENT). 62% 1874 1921 66% 1185 1361 55% 1621 1866 HWHGU54 695695 131 HMMER 2.1.1 PFAM: Serpins (serine protease PF00079 501.1 277 1377 inhibitors) HWHGU54 695695 131 WUblastx.64 (Q9CQ32) 4632419J12RIK PROTEIN. Q9CQ32 61% 145 1383 HWHGU54 695695 131 WUblastx.64 (AAL99574) OL-64 protein. AAL99574 62% 145 1377 HWHGU54 695695 131 WUblastx.64 (Q9CQ32) 4632419J12RIK PROTEIN. Q9CQ32 59% 145 1383 HCEJQ69 1243825 134 HMMER 2.1.1 PFAM: Leucine Rich Repeat PF00560 116.2 573 644 HCEJQ69 1243825 134 WUblastx.64 (Q9BZR6) NOGO RECEPTOR. Q9BZR6 87% 39 1457 HCEJQ69 1243825 134 WUblastx.64 (AF283463) Nogo receptor [Homo gb|AAG53612.1|AF283463_1 87% 39 1457 sapiens] HCEJQ69 872582 136 HMMER 2.1.1 PFAM: Leucine Rich Repeat PF00560 116.2 573 644 HCEJQ69 872582 136 WUblastx.64 (Q9BZR6) NOGO RECEPTOR. Q9BZR6 90% 39 1361 HCEJQ69 609999 138 HMMER 2.1.1 PFAM: Leucine Rich Repeat PF00560 114.5 573 644 HCEJQ69 609999 138 WUblastx.64 (Q9BZR6) NOGO RECEPTOR. Q9BZR6 59% 1100 1462 85% 39 1460 HCEJQ69 609999 138 WUblastx.64 (Q9BZR6) NOGO RECEPTOR. Q9BZR6 85% 39 1364 45% 1100 1462 HT5GJ57 740767 159 WUblastx.64 (Q9NZY9) HSPC046. Q9NZY9 90% 754 1002 70% 122 799 HT5GJ57 740767 159 WUblastx.64 (Q9GZY6) CDNA FLJ11237 FIS, Q9GZY6 84% 122 856 CLONE PLACE1008531 (WBSCR5) (WBSCR15 PROT HT5GJ57 740767 159 WUblastx.64 (AX083396) unnamed protein product emb|CAC33299.1| 84% 122 856 [Homo sapiens] HT5GJ57 1299921 161 WUblastx.64 (Q9GZY6) CDNA FLJ11237 FIS, Q9GZY6 89% 105 833 CLONE PLACE1008531 (WBSCR5) (WBSCR15 PROT HPIBX03 743314 169 WUblastx.64 (Q9H5V8) CDNA: FLJ22969 FIS, Q9H5V8 98% 81 2207 CLONE KAT10759. HDPBO81 892018 174 WUblastx.64 (Q9ES58) OX2 RECEPTOR Q9ES58 57% 400 1323 PRECURSOR. 38% 265 381 HWBFY57 837478 179 HMMER 2.1.1 PFAM: Immunoglobulin domain PF00047 19.2 209 442 HWBFY57 837478 179 WUblastx.64 (AAH19814) Hypothetical 25.0 kDa AAH19814 54% 134 535 protein. HYABV21 1281466 182 HMMER 2.1.1 PFAM: Immunoglobulin domain PF00047 46 166 342 HYABV21 1281466 182 blastx.2 MMAN-g protein precursor. sp|BAB18569|BAB18569 54% 109 672 HYABV21 1213593 184 HMMER 2.1.1 PFAM: Immunoglobulin domain PF00047 46 174 350 HYABV21 1213593 184 blastx.2 MMAN-g protein precursor. sp|BAB18569|BAB18569 49% 117 686 HOHBY69 827480 186 HMMER 2.1.1 PFAM: FG-GAP repeat PF01839 223 1696 1875 HOHBY69 827480 186 WUblastx.64 (Q9UKX5) INTEGRIN ALPHA-11 ITAH_HUMAN 98% 82 3648 PRECURSOR. HOHBY69 815681 188 HMMER 2.1.1 PFAM: FG-GAP repeat PF01839 223 1698 1877 HOHBY69 815681 188 blastx.2 (AF109681) integrin alpha-11 subunit gb|AAF01258.1|AF109681_1 99% 84 3170 precursor [Homo sapiens] 99% 3172 3648 HDHMA45 902513 202 WUblastx.64 (Q9H3S3) TRANSMEMBRANE TMS5_HUMAN 91% 175 1428 PROTEASE, SERINE 5 (EC 3.4.21.—) (SP HDHMA45 812764 204 HMMER 2.1.1 PFAM: Trypsin PF00089 296.3 723 1415 HDHMA45 812764 204 WUblastx.64 (Q9H3S3) TRANSMEMBRANE TMS5_HUMAN 99% 180 1442 PROTEASE, SERINE 5 (EC 3.4.21.—) (SP HMADJ14 1099342 206 WUblastx.64 (Q9H295) DC-SPECIFIC Q9H295 99% 200 1306 TRANSMEMBRANE PROTEIN. HMADJ14 889659 208 blastx.2 (AF305068) DC-specific transmembrane gb|AAG39167.1|AF305068_1 90% 186 1019 protein [Homo sapiens] 96% 1010 1084 HMADJ14 843725 210 WUblastx.64 (Q9H295) DC-SPECIFIC Q9H295 96% 871 945 TRANSMEMBRANE PROTEIN. 90% 47 880 HMADJ14 843725 212 WUblastx.64 (Q9H295) DC-SPECIFIC Q9H295 96% 871 945 TRANSMEMBRANE PROTEIN. 90% 47 880 HMADJ14 795479 214 WUblastx.64 (Q9H295) DC-SPECIFIC Q9H295 100% 1010 1393 TRANSMEMBRANE PROTEIN. 93% 186 1067 HMADJ14 426068 216 blastx.2 (AF305068) DC-specific transmembrane gb|AAG39167.1|AF305068_1 91% 47 685 protein [Homo sapiens] 78% 675 881 96% 872 946 HEMFA84 608198 222 WUblastx.64 (Q9H6H3) CDNA: FLJ22282 FIS, Q9H6H3 100% 42 812 CLONE HRC03861. HDPPA04 904765 224 HMMER 2.1.1 PFAM: Immunoglobulin domain PF00047 19.1 373 582 HDPPA04 904765 224 WUblastx.64 (Q9BQ51) BUTYROPHILIN Q9BQ51 92% 271 1089 PRECURSOR B7-DC (PD-1-LIGAND 2 PROTEIN). HE2OA95 637595 230 WUblastx.64 (Q9UIK5) TMEFF2 PROTEIN Q9UIK5 99% 1647 1231 PRECURSOR (TRANSMEMBRANE PROTEIN TENB2) (TPEF HKABZ65 862030 232 WUblastx.64 (Q96LB9) Peptidoglycan recognition Q96LB9 90% 77 802 protein-I-alpha precursor. 39% 137 541 HKABZ65 862030 232 WUblastx.64 (Q96LB9) Peptidoglycan recognition Q96LB9 99% 77 802 protein-I-alpha precursor. 45% 137 541 HKABZ65 665424 234 WUblastx.64 (Q96LB9) Peptidoglycan recognition Q96LB9 99% 69 794 protein-I-alpha precursor. 45% 129 533

RACE Protocol for Recovery of Full-Length Genes

Partial cDNA clones can be made full-length by utilizing the rapid amplification of cDNA ends (RACE) procedure described in Frohman, M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988). A cDNA clone missing either the 5′ or 3′ end can be reconstructed to include the absent base pairs extending to the translational start or stop codon, respectively. In some cases, cDNAs are missing the start codon of translation, therefor. The following briefly describes a modification of this original 5′ RACE procedure. Poly A+ or total RNA is reverse transcribed with Superscript II (Gibco/BRL) and an antisense or complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon). The first-strand cDNA is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (XhoI, SalI and ClaI) at the 5′ end and a primer containing just these restriction sites. This double-stranded cDNA is PCR amplified for 40 cycles with the same primers as well as a nested cDNA-specific antisense primer. The PCR products are size-separated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (PROMEGA™), restriction digested with XhoI or SalI, and ligated to a plasmid such as pBLUESCRIPT™ SKII (STRATAGENE™) at XhoI and EcoRV sites. This DNA is transformed into bacteria and the plasmid clones sequenced to identify the correct protein-coding inserts. Correct 5′ ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone. Similar methods known in the art and/or commercial kits are used to amplify and recover 3′ ends.

Several quality-controlled kits are commercially available for purchase. Similar reagents and methods to those above are supplied in kit form from Gibco/BRL for both 5′ and 3′ RACE for recovery of full length genes. A second kit is available from CLONTECH™ which is a modification of a related technique, SLIC (single-stranded ligation to single-stranded cDNA), developed by Dumas et al., Nucleic Acids Res., 19:5227-32 (1991). The major differences in procedure are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.

An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNA library double-stranded DNA. An asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.

RNA Ligase Protocol for Generating the 5′ or 3′ End Sequences to Obtain Full Length Genes

Once a gene of interest is identified, several methods are available for the identification of the 5′ or 3′ portions of the gene which may not be present in the original cDNA plasmid. These methods include, but are not limited to, filter probing, clone enrichment using specific probes and protocols similar and identical to 5′ and 3′ RACE. While the full length gene may be present in the library and can be identified by probing, a useful method for generating the 5′ or 3′ end is to use the existing sequence information from the original cDNA to generate the missing information. A method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length gene. (This method was published by Fromont-Racine et al., Nucleic Acids Res., 21(7):1683-1684 (1993)). Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest, is used to PCR amplify the 5′ portion of the desired full length gene which may then be sequenced and used to generate the full length gene. This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction can then be used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the relevant gene.

The present invention also relates to vectors or plasmids which include such DNA sequences, as well as the use of the DNA sequences. The material deposited with the ATCC™ (e.g., as described in columns 2 and 3 of Table 1A, and/or as set forth in Table 6 or 7) is a mixture of cDNA clones derived from a variety of human tissue and cloned in either a plasmid vector or a phage vector, as described, for example, in Tables 1A and 7. These deposits are referred to as “the deposits” herein. The tissues from which some of the clones were derived are listed in Table 7, and the vector in which the corresponding cDNA is contained is also indicated in Table 7. The deposited material includes cDNA clones corresponding to SEQ ID NO:X described, for example, in Table 1A (ATCC™ Deposit No:Z). A clone which is isolatable from the ATCC™ Deposits by use of a sequence listed as SEQ ID NO:X, may include the entire coding region of a human gene or in other cases such clone may include a substantial portion of the coding region of a human gene. Furthermore, although the sequence listing may in some instances list only a portion of the DNA sequence in a clone included in the ATCC™ Deposits, it is well within the ability of one skilled in the art to sequence the DNA included in a clone contained in the ATCC™ Deposits by use of a sequence (or portion thereof) described in, for example Tables 1A, 1B.1, 1B.2, or 2, by procedures hereinafter further described, and others apparent to those skilled in the art.

Also provided in Tables 1A and 7 is the name of the vector which contains the cDNA clone. Each vector is routinely used in the art. The following additional information is provided for convenience.

Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBLUESCRIPT™ (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from STRATAGENE™ Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Phagemid pBS may be excised from the Lambda Zap and Uni-Zap XR vectors, and phagemid pBK may be excised from the Zap Express vector. Both phagemids may be transformed into E. coli strain XL-1 Blue, also available from STRATAGENE™.

Vectors pSport1, pCMVSport 1.0, pCMVSport 2.0 and pCMVSport 3.0, were obtained from LIFE TECHNOLOGIES™, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from LIFE TECHNOLOGIES™. See, for instance, Gruber, C. E., et al., Focus 15:59-(1993). Vector lafmid BA (Bento Soares, Columbia University, New York, N.Y.) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from LIFE TECHNOLOGIES™. See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991).

The present invention also relates to the genes corresponding to SEQ ID NO:X, SEQ ID NO:Y, and/or the deposited clone (ATCC™ Deposit No:Z). The corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.

Also provided in the present invention are allelic variants, orthologs, and/or species homologs. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO:X or the complement thereof, polypeptides encoded by genes corresponding to SEQ ID NO:X or the complement thereof, and/or the cDNA contained in ATCC™ Deposit No:Z, using information from the sequences disclosed herein or the clones deposited with the ATCC™. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.

The polypeptides of the invention can be prepared in any suitable manner. Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.

The polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification, such as multiple histidine residues, or an additional sequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified. A recombinantly produced version of a polypeptide, including the secreted polypeptide, can be substantially purified using techniques described herein or otherwise known in the art, such as, for example, by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988). Polypeptides of the invention also can be purified from natural, synthetic or recombinant sources using techniques described herein or otherwise known in the art, such as, for example, antibodies of the invention raised against the polypeptides of the present invention in methods which are well known in the art.

The present invention provides a polynucleotide comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA sequence contained in ATCC™ Deposit No:Z. The present invention also provides a polypeptide comprising, or alternatively, consisting of, the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X or a complement thereof, a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z, and/or the polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C. Polynucleotides encoding a polypeptide comprising, or alternatively consisting of the polypeptide sequence of SEQ ID NO:Y, a polypeptide encoded by SEQ ID NO:X, a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z, and/or a polypeptide sequence encoded by a nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C are also encompassed by the invention. The present invention further encompasses a polynucleotide comprising, or alternatively consisting of, the complement of the nucleic acid sequence of SEQ ID NO:X, a nucleic acid sequence encoding a polypeptide encoded by the complement of the nucleic acid sequence of SEQ ID NO:X, and/or the cDNA contained in ATCC™ Deposit No:Z.

Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C, or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in column 6 of Table 1C, or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.

Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1), or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1) and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.

Further, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2), or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2), or any combination thereof. In further embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in column 6 of Table 1C which correspond to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (See Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.

Moreover, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of Table 1C column 6, or any combination thereof. Additional, representative examples of polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in the same row of Table 1C column 6, or any combination thereof. In preferred embodiments, the polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the complementary strand(s) of the sequences delineated in the same row of Table 1C column 6, wherein sequentially delineated sequences in the table (i.e. corresponding to those exons located closest to each other) are directly contiguous in a 5′ to 3′ orientation. In further embodiments, above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1C, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1C, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated in the same row of Table 1C, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1C, column 2) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in column 6 of Table 1C which correspond to the same Clone ID (see Table 1C, column 1), and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B.1, 1B.2, or 1C) or fragments or variants thereof. In preferred embodiments, the delineated sequence(s) and polynucleotide sequence of SEQ ID NO:X correspond to the same Clone ID. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.

In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more of the sequences delineated in the same row of column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B.1, 1B.2, or 1C) or fragments or variants thereof. In preferred embodiments, the delineated sequence(s) and polynucleotide sequence of SEQ ID NO:X correspond to the same row of column 6 of Table 1C. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.

In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of the sequence of SEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1C are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.

In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1C are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides, are also encompassed by the invention.

In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.

In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 corresponding to the same Clone ID (see Table 1C, column 1) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.

In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence in which the 3′ 10 polynucleotides of one sequence in column 6 corresponding to the same contig sequence identifier SEQ ID NO:X (see Table 1C, column 2) are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.

In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 corresponding to the same row are directly contiguous. In preferred embodiments, the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C is directly contiguous with the 5′ 10 polynucleotides of the next sequential exon delineated in Table 1C, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.

Table 3

Many polynucleotide sequences, such as EST sequences, are publicly available and accessible through sequence databases and may have been publicly available prior to conception of the present invention. Preferably, such related polynucleotides are specifically excluded from the scope of the present invention. Accordingly, for each contig sequence (SEQ ID NO:X) listed in the fifth column of Table 1A and/or in fourth column of Tables 1B.1 or 1B.2, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 and the final nucleotide minus 15 of SEQ ID NO:X, b is an integer of 15 to the final nucleotide of SEQ ID NO:X, where both a and b correspond to the positions of nucleotide residues shown in SEQ ID NO:X, and where b is greater than or equal to a+14. More specifically, preferably excluded are one or more polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a and b are integers as defined in columns 4 and 5, respectively, of Table 3. In specific embodiments, the polynucleotides of the invention do not consist of at least one, two, three, four, five, ten, or more of the specific polynucleotide sequences referenced by the Genbank Accession No. as disclosed in column 6 of Table 3 (including for example, published sequence in connection with a particular BAC clone). In further embodiments, preferably excluded from the invention are the specific polynucleotide sequence(s) contained in the clones corresponding to at least one, two, three, four, five, ten, or more of the available material having the accession numbers identified in the sixth column of this Table (including for example, the actual sequence contained in an identified BAC clone). In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety. Table 3, from U.S. patent application Ser. No. 10/472,532, filed Sep. 20, 2003, is herein incorporated by reference. Table 3 in priority Application No. PCT/US02/09785, filed Mar. 19, 2002, which corresponds to Publication No. WO02/95010, published Nov. 28, 2002 (e.g., pages 643 to 798 of Publication No. WO02/95010) is incorporated by reference herein in its entirety.

TABLE 3 SEQ EST Disclaimer cDNA ID Contig Range Range Clone ID NO: X ID: of a of b Accession Numbers HNFIP24 1 351534 H04128, AA099288, AA101983 HETBY74 7 361396 R23770, R26913, R32172, R32216, R38317, R39395, R63720, R63767, R70906, R70940, R70993, R77169, R82584, R82585, H01942, H04495, H21696, H21906, H97740, H99748, N62092, N95055, W38893, W93362, W93394, W93606, AA016203, AA016243, AA076610, AA079633, AA079807, AA086218, AA158761, AA159815, AA159816, AA159710, AA159711, AA159565, AA160381, AA186897, AA188520 HTEEB42 9 206980 1-1008 15-1022 AL522795, AA725566, AI421450, AL522796, AI199779, AA406389, AA912674, AW022835, AI952846, AI123727, BE218057, AW022646, N90730, BF846982, BF845761, AI652914, BF056970, AW020783, AI312805, AW393829, AI017553, AW393887, AW474261, AW264246, BF848293, AI366088, AI418268, T89217, AI052637, AW082343, BF221504, AW593293, AA865038, AI201753, BF091146, AI140139, AA987434, AA410345, BF846977, BF846980, AW900593, BF932982, BF932991, AW865421, AW136481, AI650503, AI432092, T89127, AA974715, AW261924, BE938414, AF255910.1, AY016009.1, AP001694.1, AP000087.1, AP000225.1, AP000226.1, AP000086.1, AP000223.1. HTEEB42 9 206980 1-1008 15-1022 AL522795, AA725566, AI421450, AL522796, AI199779, AA406389, AA912674, AW022835, AI952846, AI123727, BE218057, AW022646, N90730, BF846982, BF845761, AI652914, BF056970, AW020783, AI312805, AW393829, AI017553, AW393887, AW474261, AW264246, BF848293, AI366088, AI418268, T89217, AI052637, AW082343, BF221504, AW593293, AA865038, AI201753, BF091146, AI140139, AA987434, AA410345, BF846977, BF846980, AW900593, BF932982, BF932991, AW865421, AW136481, AI650503, AI432092, T89127, AA974715, AW261924, BE938414, AF255910, AP001694, AP000087, AF255911, AX036060, AP000225, AP000226, AP000086, and AP000223. HEQCC55 15 1352368 1-986  15-1000 BF344112, BE275042, BE275057, BE563860, BE876310, BE880309, BE910481, BE733268, BF526321, BG169508, BE876530, BE878582, BE384718, BE562629, BG170369, BG170840, BE304867, AI492143, AI768116, BE876850, BG164974, BF338794, BE274902, AA149044, AW204761, AI768403, AI221536, BE395085, BG032898, AW172990, BG248912, AW051452, AW262030, BF114971, AI800959, AW338518, AI827127, AI568941, AI761510, F25336, AI313436, AI086734, N41733, AA994944, AW938942, BF063028, AW628237, AI219327, AW859588, AI718198, BF108787, AI004154, AI864228, BF197631, AI767239, AW001699, AI796303, T56712, AA576558, AI911799, AW149867, AI470703, AA149043, BG236779, AI470484, N83862, BG249788, AA631934, AI270718, AA610401, AI813825, AI401800, AI358289, AI799902, H95227, AW166567, AI264959, AI611273, R48167, BE730123, AI867518, AW815436, AW815444, BF338447, AW391445, AW815697, AI167239, AA970894, AW815514, T74424, BE076102, AW815626, BE076131, BG168390, BG169301, AI910684, AI189491, BE075966, AW843216, AA386018, BE076032, AA873480, R33355, T74049, BF338531, BF734770, AI919408, AI858303, AI701259, D29265, BF331419, BF982285, BE042437, AA996249, BG002211, AW374913, BC002718.1, AF191148.1, AB035480.1, AC004643.1, AB035481.1. HEMCM42 15 1352150 1-986  15-1000 BF344112, BE275042, BE275057, BE563860, BE876310, BE880309, BE910481, BE733268, BF526321, BG169508, BE876530, BE878582, BE384718, BE562629, BG170369, BG170840, BE304867, AI492143, AI768116, BE876850, BG164974, BF338794, BE274902, AA149044, AW204761, AI768403, AI221536, BE395085, BG032898, AW172990, BG248912, AW051452, AW262030, BF114971, AI800959, AW338518, AI827127, AI568941, AI761510, F25336, AI313436, AI086734, N41733, AA994944, AW938942, BF063028, AW628237, AI219327, AW859588, AI718198, BF108787, AI004154, AI864228, BF197631, AI767239, AW001699, AI796303, T56712, AA576558, AI911799, AW149867, AI470703, AA149043, BG236779, AI470484, N83862, BG249788, AA631934, AI270718, AA610401, AI813825, AI401800, AI358289, AI799902, H95227, AW166567, AI264959, AI611273, R48167, BE730123, AI867518, AW815436, AW815444, BF338447, AW391445, AW815697, AI167239, AA970894, AW815514, T74424, BE076102, AW815626, BE076131, BG168390, BG169301, AI910684, AI189491, BE075966, AW843216, AA386018, BE076032, AA873480, R33355, T74049, BF338531, BF734770, AI919408, AI858303, AI701259, D29265, BF331419, BF982285, BE042437, AA996249, BG002211, AW374913, BC002718.1, AF191148.1, AB035480.1, AC004643.1, AB035481.1. HEMAE80 20 409495 T71556, T90634, T82005, T83161, H57113, H61567, AA233071 HEMAE80 22 1310948 1-1078 15-1092 AI742654, AA461037, BF949730, AA460463, AI571360, AA701618, AI127309, AI633508, AA233071, AA448648, AA448744, AA897786, BF477408, AI206655, T82005, AI589742, T71556, T83161, T90634, H61567, H57113, H60996, AV707024, AV725948, AV701724, AV727990, AV706677, AV702026, AV729131, AV703989, AW949478, AV728436, AV727314, AV727935, AW949477, AV728518, AV728523, AV725134, AV656283, AV696791, AV698429, AV684604, AV692972, AV685688, AV689800, AV693005, AV656478, AV702516, AW964421, AV702266, AV709660, AV687909, AV656903, AV661704, AV697196, AV655280, AV659322, AV709314, AV708381, AV660728, AV691080, AV703169, AV659536, AV706721, AV729220, AV687035, AV655096, AV706223, AV726816, AV695545, AV708025, AV707933, AV708980, AV692691, AV701914, AV708992, AV726103, AV727029, AV728733, AV727100, AV705280, AV686064, AW960720, AV686060, AV703515, AV703472, AW955900, AW951263, AW967047, AV704553, AV704785, AW953797, AV692600, AV651955, AV725208, AW952751, AV727238, AV709880, AV696866, AV725920, AV693523, AV706342, AV725826, AW949353, AV654908, AV728546, AV692345, AV726584, AV698290, AV698609, AV725582, AV649758, AV708438, AV696106, AV689111, AV728157, AV708893, AV704217, AV702280, AV725031, AW951280, AV696931, AV726624, AV709604, AV652001, AV701946, AV704955, AV702117, AW954248, AV701707, AV707753, AV704269, AV702832, AV701560, AV705693, AV708704, AV726738, AV703495, AW964095, AV702772, AV726156, AV697288, AV652290, AV648263, AV647789, AV727787, AV660608, AV698545, AW955053, AV725497, AW952410, AV699089, AW950443, AV654035, AV706854, AV728997, AV656256, AW960601, AW956199, AW952403, AV704234, AV729378, AW952183, AV705159, AW959806, AV709407, AV703494, AV725633, AW956075, AV645936, AV709587, AW955723, AV658084, AW962384, AV650315, AV659389, AV697880, AV727613, AV726010, AV660258, AV708109, AW956474, AV659294, AV703146, AV725745, AV728148, AW951239, AW020543, AV726590, AV653353, AV654070, AW951281, AV706219, AV702385, AV658275, AW949802, AV702790, AV703669, AV707979, AV726194, AV727003, AV709580, AV702372, AV708786, AV659547, AW957517, AW959543, AV727526, AV705416, AV651920, AV725618, AW954439, AV707541, AV725577, AV725033, AV728924, AF193766.1, AF274956.1, Z59544.1, Y08991.1, AF217994.1. HRDFB85 28 411020 1-1691 15-1705 AL526995, AU133713, AU122264, AU133716, AU133898, AU133901, AU121966, AU141449, BE792903, BE786350, AU133817, AU136746, AU133887, AU121817, BG254161, AU133655, AU133578, AU121969, AU133752, BE907881, BG260814, AU133916, AU136516, BG179180, AU134354, BF038418, BF036516, BF037422, AU138430, BE734836, BE873066, AU133630, BE875603, AW844114, AU122380, AU134380, BE616872, BE871117, BE880804, BE871864, AU134369, BE902820, BG256045, BE536853, AU133618, BE304643, BE787818, AU147972, AU138507, AA700004, AI927220, AW170580, W74492, BF446247, BE812430, AU147690, BE870378, AI991311, AW935041, AA523290, AI859845, BF681403, AU136509, AI081052, AU155374, AA535079, AU159009, AI400364, AU147886, AI335984, AU155390, BG254485, AW193221, AW874638, AU155077, BE564185, AW170345, AA622540, AU155064, AI273767, AA522795, AW474974, AW770097, BF445088, AW168283, AI188508, AI559433, AI420481, AI246782, AU157642, BF182989, BF825787, AW815382, BF110949, AW662920, AI928146, AA157892, BG000738, AU133955, AA314960, AW815398, AI281336, AU155007, BE740807, AU155054, BF447199, AU154870, AA838633, AA844471, AW589514, AI401064, AW844815, AW815552, AI949231, AU154943, AI911649, AI268908, AI874198, AI186144, AI819846, BF742353, AW193220, AI863584, AL036495, BE930559, AA149417, AW168206, AA565989, W79089, BE819070, AA506616, AI564546, BF987328, BF435486, BE903940, AA434123, AA149738, W02467, AW194453, AA948146, AU155067, AL526954, C06165, AI276313, BG002911, AI963847, BG254948, AI874344, BF476318, BF987337, AI660464, AI567796, BE745379, BE673504, BF446395, AW167101, BF990584, AA434059, AI739607, AI560666, AI280032, AI961910, R48300, AW009339, BE840036, AA551656, BE840048, AW167849, AW513272, BF001751, AI346572, AI923100, AI005290, AI091394, H93341, AI588982, AI819915, AI950029, AI991855, AI347074, AI347076, AI660868, AA295491, BE930558, AW874110, AI682624, AI348165, AI949885, BF001991, AW374558, AW516096, AI347071, AU154878, AW014104, AI343369, AI860565, AW167111, AI222884, AI861959, AW194388, BE840202, BF109128, AI283186, AI347501, AI305833, AI031766, AI346386, AI346944, AW189088, AI032425, H27323, AI283162, BE048493, BF569270, AI347072, AW510456, BE840244, BF001523, BF111772, AI214245, AI346606, AI743195, BE840045, AW015201, AI347060, AI346569, AW572261, AW275383, AI281140, AI346475, AI743978, AI274133, AI738882, AI347930, AI273374, AI738627, AI991114, AI097004, AI144005, U46417, AI304544, AA157596, AI281141, AA569935, AI274318, AI285074, AI346274, AI336454, AI346908, AI339875, AK026651, AB000712, D88492, AX014904, AB000714, AF007189, AF095905, AJ011656, M74067, AJ130941, AC004643, AJ249735, R12121, T96099, R05961, R05962, R36883, R48403, R50075, R50076, H13937, H27324, H27350, H44304, H93844, N72688, W21446, AA149303, AA149402. HDTAW95 38 412472 1-1274 15-1288 AL532456, BE896915, BE387335, AW071610, AA584310, AI762109, BF691507, AW994682, AI085616, BE550475, AI422726, AW316980, AI809642, AI743774, BF000103, AA482398, BE551643, AI359844, BE550264, AI379443, AA406425, AA482544, AW518948, BE221566, AW276370, AI656907, AA410434, AI380885, AW236626, BE169370, AI963298, AA857920, AW519014, AI352209, AV649395, AI469175, AI920963, AI631574, AI700002, AI750892, AI218433, AA335862, C01758, AI760411, BF432104, AA335551, AW594188, AW084890, AA723450, AI653051, AA738416, AI074769, AI081084, AA974239, AA969841, AW137377, AI239604, AI391517, AW594685, AW518917, BF000821, AI370649, AW662493, R46762, BE843536, AA507081, BF507778, AW630561, AI750893, AW837990, R46857, AW608087, AW838003, AI074870, AI369474, BE504418, D62262, BE698661, F13673, AW627356, D79314, AI418593, AI337269, AW627526, AA588673, BF573143. HEMCV19 46 423219 R39576, R39644, R55519, R55520, H25585, H25630, H42497, H43485, R95168, H73675, H73419, H80718, H80719, W95391, W95348, AA034079, AA044081, AA187305, AA187096 HEMCV19 48 1352162 1-722  15-736  BE614883, BE873075, BG025158, BE253565, BE875979, BE395207, AW954217, BE734057, BE789549, BF529722, BE614806, BE738333, BE785505, AA044211, BF340317, AW238972, AW406916, R95168, BF737302, BF812968, BF738993, AA075901, BF792671, BG012029, AW796084, H25630, BE876237, BE183450, BF901660, BE810793, H43485, AA296837, H80718, AA287470, AA298795, AA296826, AA296696, AI342703, AA296869, AA218811, AW802996, AA034079, BE737872, H73675, R55519, W95348, BF331433, AV743953, BF082160, BF868942, BF809994, BE775061, C00212, AL532494, BF997411, R39644, BE828356, BF804848, AA806231, AL532493, BF799156, BF895320, AA187096, AA297863, BF245896, BF905334, N86960, T10507, BE904822, BF931829, BF829581, BE140100, AI819836, BE396147, BF747982, BF056207, BF763569, BF807379, AA465105, BF811185, BF446055, AA583464, AA485829, AI582284, AA983595, BF771777, BF750444, AA523623, BF771828, AI952620, AA044081, BF736987, AI346295, BF978464, BF248485, AA912088, AW512207, AI028038, AI066489, AI424071, AI521467, AA699982, AI358167, AA722634, H42497, AC002390.1, AF177940. 1. HETBX14 61 806447 1-1278 15-1292 BE867930, BE219655, BF476474, AW511566, AI521607, AI217150, AI183346, AI283289, AW000834, AA436049, AA435952, AA532717, AA403004, BE673570, AI624187, BE898804, W60282, AI913780, W60374, AA402971, AA477283, BF679282, AA412318, AA477282, AW969530, BE150851, AA482081, BF222155, AA402028, BE646241, BF677072, AA514646, BE150769, AA503821, AI611257, AI659265, BE828491, BF515767, AW578446, AW166796, AI874153, BF573830, AW874467, AB041036, AB012917, AX016289, AX016287, AB013730, AC011473, AF164623, AF243527, AR060847, AB016226, AF135025, AC011473, AC011473. HETBX14 65 422659 W60282 HLHSK94 70 422828 R55809, H83295, N92239, W37154, W38638, N90902, AA017680, AA040604, AA040705 HLHSK94 72 1307727 1-1775 15-1789 AW956492, BF352284, BE709448, W29010, AV751953, AI829559, BE179448, AI571060, AI083491, AA905071, AW118125, AI049799, W22553, N90902, W27632, AI273588, AI890622, W27896, AW195777, W22119, BF844755, AA040604, W23268, AW269932, W38638, W37154, AA904910, W27944, R55894, W27681, AW607334, AA337059, AW367713, AW607165, BF351195, AW966017, AA298658, R55809, C02576, BE163743, AI376671, AA364393, AV746728, AI393483, W23093, BE708746, W28670, W27851, BF844715, H83295, BF437730, N92239, AI194027, W27371, D81988, BE163611, BE163731, C14616, C14877, AA040705, AA897696, AV747434, BF850779, AA017680, H83294, AW974810, AF122922.1. HLHFP03 76 460467 1-599  15-613  H46196, AI421986, H19572, H46195, BF947135, H19490, BF738481, BF994257, BF127477, AW139949, BF947011, AF141377, AF169202, AC011976, and AC011976. HLHFP03 78 460467 1-599  15-613  H46196, AI421986, H19572, H46195, BF947135, H19490, BF738481, BF994257, BF127477, AW139949, BF947011, AF321824. 1. HHTLF25 83 461438 1-683  15-697  AA481924, BF343628, AI276798, BE858514, BF915546, BG058647, BF917552, AI299346, N41026, BF914451, AA989053, W60864, BF915075, AI423526, BF106006, AI289858, AA746220, BF915128, AI306602, AW015647, AA633118, AI207255, BF913974, AI301688, W92376, AI139176, AA971275, AA480109, H12338, BF912934, AA865668, BF901361, F30553, AW975896, AA991168, AI302882, BF915115, AA729941, AA627378, AA865651, AW607348, H39980, AA729534, T55959, T57206, AW607175, W60940, BE155729, AI880682, AW383808, BG058709, AW383055, AW383057, BE154544, AW383016, AW383047, AW383871, AW383051, BF901355, AW383000, AI919456, BE154555, AW383784, BF914191, F32872, AI017727, AA974881, BE154538, AW383009, BF092099, AI243983, AA991170, R49835, R49793, AA318120, BF893642, W74783, AW382999, AV712713, AW579628, AW382994, H12392, AW372144, AW372157, AW383836, T52100, AW372154, AW383822, AW383837, AW579627, AW383817, AW372166, BF881098, AW382997, D20493, AW372161, AW383865, AA918360, N47127, AW579992, AA937670, AW579601, AW579998, AU076484, AI245273, BF831159, AA664094, AA878598, AA865673, AI807718, AA937805, BF350664, AI525220, AD000833.1, AF019563.1, AF019562.1, AJ010098.1, AD000864.1, X78928.1, AF072845.1. HHTLF25 83 461438 1-683  15-697  AA481924, BF343628, AI276798, BE858514, BF915546, BG058647, BF917552, AI299346, N41026, BF914451, AA989053, W60864, BF915075, AI423526, BF106006, AI289858, AA746220, BF915128, AI306602, AW015647, AA633118, AI207255, BF913974, AI301688, W92376, AI139176, AA971275, AA480109, H12338, BF912934, AA865668, BF901361, F30553, AW975896, AA991168, AI302882, BF915115, AA729941, AA627378, AA865651, AW607348, H39980, AA729534, T55959, T57206, AW607175, W60940, BE155729, AI880682, AW383808, BG058709, AW383055, AW383057, BE154544, AW383016, AW383047, AW383871, AW383051, BF901355, AW383000, AI919456, BE154555, AW383784, BF914191, F32872, AI017727, AA974881, BE154538, AW383009, BF092099, AI243983, AA991170, R49835, R49793, AA318120, BF893642, W74783, AW382999, AV712713, AW579628, AW382994, H12392, AW372144, AW372157, AW383836, T52100, AW372154, AW383822, AW383837, AW579627, AW383817, AW372166, BF881098, AW382997, D20493, AW372161, AW383865, AA918360, N47127, AW579992, AA937670, AW579601, AW579998, AU076484, AI245273, BF831159, AA664094, AA878598, AA865673, AI807718, AA937805, BF350664, AI525220, AD000833, AF019563, AF019562, AJ010098, A83704, AF152021, AF247680, AD000864, AF285446, 125670, X78928, and AF072845. HTADX17 90 457172 1-1126 15-1140 AA446344, AA612751, AA298785, AA298780, AA298784, AA446524, AA298781, AA381170, AL357565, and AL357565. HTADX17 92 753289 1-1133 15-1147 AA446344, AA612751, AA298785, AA298780, AA298784, AA446524, AA298781, AA381170. HJACG02 104 1307789 1-561  15-575  AA311223, BF002026, N41594, N30820, BF982046, AI829327, BE047833, AI457369, AW071417, BF968205, AI340627, R36271, AL036980, BF061283, BG168549, AW022682, BG034550, AV682418, AL047042, BF343172, BG113299, AW020693, BF751308, AI452560, AI690748, AI349645, AW946806, AI340511, BF924882, AW074869, AW196299, AL038445, BE781369, AW302992, BG110684, BE887488, AL514193, AI310575, BG164558, AI340533, AI349957, AI433384, BF680133, AV715560, AI309401, AI345005, BG163618, AI343112, AV743962, AI826225, AI811785, AI494201, AW054931, AW268302, AW301300, AI349598, BF672397, AW072719, AW075207, BF526020, AV741327, AI345735, BG036846, AI697243, BE536058, AW193134, AI889147, BF904189, BE910373, AI500077, AA225339, BE138712, AI307210, BG033723, AI589267, AI269862, BE885353, AI313320, BG058150, BE886728, AW827106, BF527014, AI313352, BG110517, AL039086, AW079336, AI251434, AI274728, BF868928, AI524780, AI589947, AV682724, AI439717, AI312146, AI312339, AI814087, AI345745, AL036925, AI345258, AI932638, AI470651, AL036857, AW050578, AW196105, AV682227, AI306705, AW269097, AI620639, AI611348, AW090393, AL042628, AW152469, AA833760, BG256090, AI866798, AW074993, AI567351, AI431424, AI349614, AI311604, AW105601, BE966990, AL044207, AW167918, AI611738, AW169604, AW268253, AI862144, AI567612, BE886827, BF793308, AI890806, AI349256, AL036664, AI554821, AI312152, AI955906, AI336495, BF970768, BF885000, AW075084, AL120854, BE895585, AI950664, BE897632, BE964078, BF872670, AW022699, AI349937, AL036923, AW089572, AI334884, AI307543, AW151138, AW071412, BF885081, AI307708, AI312325, AI500659, AI868204, AI340659, BF816037, AI280655, AI612885, BF092710, AW302965, BF339322, AI334930, AI309443, AV699211, AV734185, AI307520, AI445237, AV724373, AI590423, AV756798, AI345739, AI889168, AI440263, AW117743, AI312143, AW673635, AW806761, AI343037, AV708834, AI434256, AI312428, W33163, BG109270, BE966829, AI349955, AW075093, AI371228, BE548914, AW827206, AI348897, AA427700, AI306613, AI312357, AI335426, AI348777, AI308032, AI569583, AI687127, BG249582, AI783997, BG030364, BG104820, AW161579, AI627988, AI344785, BG113662, BE971716, BF970449, AL079963, AL036718, BE047852, BE785868, AI207454, AI382670, AW020095, AI874166, AL036901, BE047952, AI670009, BG180996, BF970990, BF526262, BG027280, AL036274, BF061286, AI497733, AL041150, AI288285, AI890507, BG026428, AW827115, AW268964, AI343091, AI318280, AI567582, BG165051, AI554245, BE963035, BE138658, BG260037, AI310582, BG032208, BF344691, BE885490, AF352730.1, AF205952.1, AF323081.1, AK024538.1, X53587.1, AL512765.1, AL050393.1, AK025254.1, AF090901.1, AK026542.1, AL136787.1, AK026597.1, BC006525.1, AF218031.1, BC001963.1, BC007326.1, AB055366.1, AK027213.1, AL389939.1, AK026528.1, AK026855.1, AL122110.1, BC008780.1, AF090943.1, AL133098.1, AL136799.1, BC008070.1, BC003687.1, AK024524.1, AF091084.1, AL049466.1, AK025967.1, AK026480.1, BC002839.1, BC006807.1, AL136789.1, AL157482.1, AL117394.1, AK025391.1, AL137560.1, AY034001.1, AK025349.1, AF125948.1, AL359615.1, AJ242859.1, AK025906.1, AL136915.1, AL110221.1, AL050092.1, AL512718.1, AB056427.1, AK027146.1, AB060825.1, AL133075.1, AK025484.1, AK000391.1, AF056191.1, AB051158.1, AK026583.1, AB060908.1, AL133557.1, AL117457.1, AL110225.1, AK026526.1, AL080159.1, AL137550.1, AK026629.1, AL133640.1, AB063046.1, AF217987.1, AK026452.1, U42766.1, AL133606.1, AK026534.1, AL133067.1, AK024588.1, AB047615.1, AB048954.1, AK027182.1, AL049464.1, AL133113.1, AL133560.1, AL137521.1, BC008983.1, AB060912.1, AF097996.1, AK026353.1, BC008899.1, BC001967.1, AK026959.1, AL137459.1, AB060839.1, BC003548.1, AF090900.1, AL050116.1, AB052200.1, AL133016.1, AL359620.1, AB060214.1, AF057300.1, AF057299.1, X72889.1, AK000618.1, BC002643.1, BC008417.1, AK026164.1, AK026506.1, AK026741.1, AL359618.1, AL442082.1, BC007199.1, AK025465.1, U91329.1, L19437.2, AL049314.1, AB056421.1, BC004958.1, AK027164.1, AB056809.1, AL162062.1, AL050149.1, AL389982.1, AL137463.1, AL122123.1, AF230496.1, AL162002.1, AB048964.1, AL133080.1, AL390154.1, BC006164.1, AK000137.1, AK026762.1, AL117460.1, AK026630.1, AL512689.1, AL512719.1, AL050108.1, AK026593.1, AB063100.1, S61953.1, AL049300.1, AL049452.1, BC005151.1, AK000647.1, BC001045.1, AB063079.1, BC002733.1, AL136893.1, BC002342.1, AK025383.1, BC004556.1, BC009284.1, AL080086.1, BC005678.1, AF078844.1, AF026816.2, AL136928.1, AL512750.1, BC003627.1, Y16645.1, AB062942.1, AF051325.1, AL512754.1, BC006103.1, U58996.2, AB060863.1, AL136844.1, AK000212.1, AL080074.1, AL359601.1, AF262032.1, AK026600.1, AL136864.1, U80742.1, AL136845.1, AL122093.1, BC008893.1, AL136892.1, BC006201.1, AL117435.1, BC008365.1, AL110280.1, AF061943.1, AL136749.1, AK027868.1, AB019565.1, AF162270.1, BC008382.1, AL133104.1, AF003737.1, AF353396.1, AL137557.1, AK000445.1, BC008284.1, AK000432.1, AF218014.1, AL136786.1, AB050510.1, AK024601.1, AL117583.1, AL137648.1, AB060929.1, AK025491.1, AL117585.1, AL122098.1, AK025573.1, AF219137.1, AF090903.1, AF125949.1, AF260566.1, AL050146.1, AF111847.1, AL442072.1, AB062978.1, AJ006417.1, AL122121.1, AJ012755.1, AL136784.1, AF104032.1, BC008488.1, AL080060.1, AB055315.1, AL137478.1, AB047887.1, AF132676.1, AF061836.1, AF183393.1, AK027204.1, AL080127.1, AK026647.1, AK027116.1, AL096744.1, AB055361.1, AL353940.1, BC008387.1, AL137556.1, X82434.1, AF090934.1, AL122049.1, AL162008.1, AB063070.1, AL122050.1, AL162003.1, AL137271.1, BC005168.1, AB048974.1, AK025798.1, AB055368.1, S78214.1, AK000486.1. HKGAJ54 107 498303 1-1332 15-1346 H97115, AA130346, and AA193462. HKGAJ54 109 1300770 1-1318 15-1332 AA757140, BE301253, AL528730, AI761849, AL520073, BG150431, AI288886, BE301262, AW380873, BE018216, AA227523, AA227644, AI224842, AI417635, AI094181, H97115, AA193462, AW371297, AL039859, AA262346, AW957515, AA577940, AI435978, AA373648, AW381560, AW380920, H96998, AI240420, AA130346, W88873, W90542, AA001144, AW381554, AF074684, AA253322, BF083570, AA193344, AL520074, BE173261, AA368803, AI718525, AA831401, AA741509, AI741117, AK027852.1, BC008993.1. HE8CH92 118 609866 1-1268 15-1282 BF338867, AI862534, AA861640, AI149724, AA400490, AA759080, AA400536, AI218853, H52956, BE550640, AI476417, AW274868, BE552351, H53024, AA843555, H53025, AA936598, AW516351, T81709, AL079515, AW292593, AI280269, AA629002, AL079514. HSVAK93 120 1352228 1-1226 15-1240 BF338867, AI862534, AI149724, AA400536, AA400490, AA861640, AA759080, H52956, AI218853, H53024, BE550640, AW274868, BE552351, AI476417, H53025, AA843555, AA936598, AW516351, T81709, AL079515, AW292593, AL079514, AI280269, AA629002, BC008988.1, AC008491.6, AC010315.6. HSDEK49 125 1352253 1-1768 15-1782 AL513706, AL513705, AV700980, BF343961, AV710516, AV716397, AV715849, BF351156, AV717025, AW071975, AI922669, AI129815, BF106386, AA702864, W32947, AV690218, AV685715, AV693576, AV686846, AV695322, AV697709, BF924861, AI168499, AI343825, AA627735, AI554367, AI335089, AV697729, AI290781, AA875852, AA442570, AV686969, AV698914, AA486920, AI357884, AI088635, W79882, R39812, AV683817, BF932594, W17367, N78991, AA972857, R62969, R59135, AW961380, R56601, BE857524, R66262, W74268, AA436814, AA813538, H05057, AA133776, Z43556, R14044, R81029, T48889, AA228697, R56602, AA142932, R63023, Z39624, F02373, AA993978, R66723, R67603, R59136, R80928, AA133775, AW874480, T48888, AA228698, AA368546, BF525711, AA115592, AA328299, AA486747, BG001652, AJ132502.1, AL034397.1. HWBAO62 127 838164 1-1889 15-1903 AI683471, AI792952. HWHGU54 131 695695 1-1431 15-1445 AA458648, BE140448, AA455546, AL132708.3, AL132990.3. HWHGU54 131 695695 1-1431 15-1445 AA458648, BE140448, AA455546, AL132708, and AL132990. HCEJQ69 134 1243825 1-1763 15-1777 BE889609, BF183110, BG248693, AI621234, AA452195, AI821464, AI821685, AI820935, AI745288, AI655244, AA436362, BF432211, AW770404, BF196926, AA427404, H08678, AA410412, H61387, AI432244, AA428418, H08677, AA410413, AA410253, H14737, BF351660, BG170733, H62405, AI968569, AW002476, AW166810, BE350305, BF530203, H09849, AW748526, AF283463.1, AC058790.14, AC007663.29, AC006549.28, AB045987.1. HCEJQ69 134 1243825 1-1763 15-1777 BE889609, BF183110, BG248693, AI621234, AA452195, AI821464, AI821685, AI820935, AI745288, AI655244, AA436362, BF432211, AW770404, BF196926, AA427404, H08678, AA410412, H61387, AI432244, AA428418, H08677, AA410413, AA410253, H14737, BF351660, BG170733, H62405, AI968569, AW002476, AW166810, BE350305, BF530203, H09849, AW748526, AX047642, AC058790, AC007663, AC006549, and AB045987. HT5GJ57 159 740767 1-1783 15-1797 AW574516, BG259057, BF975647, AW575080, BF795582, BE559713, BE396519, AI521311, BE397179, AW237047, AI446257, BF238156, BF663664, AI862389, AI573063, BE513368, AW296989, AU157608, BF128855, AA811488, AA827120, AW338778, AI439638, BE560794, AI250231, BG120258, AI312540, AA633095, AV742373, AI343438, AA604586, BE269253, AI669176, AI149413, AA723128, AW403042, AA722908, W57991, AI826124, BE246032, AU138425, BE513265, AI865336, AI219708, AI589599, AW732709, H23560, AA765412, AI589912, BE560732, T61448, AW444827, AA594614, AA648496, AA361096, N34423, W58075, AV742389, N48728, AA975334, AA731435, AA166766, BE247378, AA810638, AW298682, AI919140, AW402333, AI341517, BE245894, AV743440, AI962720, BF062274, T30849, AI982795, AW405561, T25945, AA810222, AA807717, Z39117, AV756294, AV724221, BG120887, AA593214, N48658, AW083122, AA639378, H23535, AI492348, AI656821, AF045555, AK002099, AF257135, AF161531, AC005081, AF086239, AC016675, AC016675, AC005081, and AC005081. HT5GJ57 161 1299921 1-1759 15-1773 BF975647, AW574516, BG259057, BE397179, BE396519, AW575080, BF795582, BF663664, AI521311, AW237047, AI446257, AI862389, BE559713, BF128855, AI573063, BF238156, AW296989, AU157608, AA811488, AA827120, AW338778, AI439638, AI250231, AI312540, AA633095, AV742373, AI343438, BE513368, AA604586, AI669176, AI149413, AW732709, AA723128, AW403042, BE269253, BE560794, AA722908, W57991, AI826124, BE246032, BG120258, AU138425, BE513265, AI865336, AI219708, AI589599, H23560, AA765412, AI589912, T61448, BE247378, BE560732, AW444827, AA594614, AA361096, AA648496, N34423, AV743440, W58075, N48728, AV742389, AA975334, AA731435, AA166766, AA810638, AW298682, AI919140, AI341517, BE245894, AI962720, BF062274, T30849, AW402333, AI982795, T25945, AA810222, AA807717, Z39117, AV756294, N48658, AW405561, AV724221, BG120887, AA593214, AW083122, AA639378, AI492348, H23535, AI656821, AF252613.1, BC009204.1, BC001609.1, AF252611.1, AF252614.1, AK002099.1, AF257135.1, AF252612.1, AF045555.1, BC006080.1, AC005081.3, AF086239.1. HPIBX03 169 743314 1-2195 15-2209 BG163962, AU140819, BF336602, BG168220, AU147884, AW996615, BF760456, AA922764, AA770561, AA976914, AA045899, BF063943, BF851894, BF815498, AA045900, BF764764, AW769303, AW996391, AW812414, AA132854, AB033417, AA132760, AK026622, AK023834, AK026187, AC010170. HDPBO81 174 892018 1-3784 15-3798 AI393580, BE041810, AI867153, AI307279, BE568549, AX047952, AX047936, AX047940. HWBFY57 179 837478 1-1782 15-1796 AW003259, BE673705, AW236996, AW590159, BF478096, BE218330, AI802017, AI707979, AW590572, AI970192, BF062678, AW615365, AA984871, AI796486, AI365178, AW138945, AI310573, C01628, AA324946. HYABV21 182 1281466 1-2724 15-2738 AW969109, AA278948, AA677057, AA813919, AW976932, AI572979, AW294948, AW503289, AW198126, AI419925, AA810016, AA278822, AA809271, T89787, AA505047, AA804243, R09908, AW500471, R12559, AA281955, AW383680, AA767265, AW503702, AW504600, T89422, H50970, AL356276, AC024085, U85195, AE000658, AC009248, and AC004671. HOHBY69 186 827480 1-4981 15-4995 BF965934, BF358074, AW753022, BG258877, AI621089, AW851445, AI751442, AW069755, W45174, H15811, AW339880, AI752628, AI751441, AW068275, BF509527, AW604115, BF056654, H16112, AI825217, W45078, BF749563, BF770980, BF770976, U53091, AA852615, AI738528, BF770978, BF928722, BF770979, AI750918, BF365293, AW888738, AA665807, BF328395, BG013602, AA852614, BF770969, AI752385, AI752488, BF770975, BF770962, AA316377, BF770974, AI752355, BF769640, BF758396, BF758424, BF830392, AI750919, AV699874, AV700783, AW166270, AF109681, AF137378, AL359064, Z50167, Z50157. HDHMA45 202 902513 1-2170 15-2184 AL538140, AI653241, AI826089, AI967938, AW003801, BE222599, AI056603, AI085672, AI201055, AI367072, AI052212, AI631456, AI278127, BF515139, AI597622, AI825589, BF961191, N47437, AA506257, AI197773, AI040587, BF934870, BF934966, AB028140.1, AP002436.3. HMADJ14 206 1099342 1-1350 15-1364 AI268407, AW450309, AI831182, AW295136, AF305068. HMADJ14 212 843725 1-1430 15-1444 AI268407, BE162690, AI831182, AW450309, AW295136, AA380009, AF305068. HEAAL31 218 639007 AA151656, AA151652, AA151733, AA151736 HEAAL31 220 361221 T56046, T56062, T56080, T56096, T90475, T90482, T90571, T90577, T74331, T89037, R10054, R11653, R54570, H22902, H24285, H24287, H50611, H50610, H67882, H67927, N27199, N36263, N39942, N40544, N44078, N46591, N58937, N73790, N94307, N95519, N99415, W02128, W19438, W32393, W57988, W58072, W73132, W90660, N90316, AA009898, AA022614, AA022615, AA025590, AA081613, AA084503, AA085189 HEMFA84 222 608198 1-971  15-985  BE743873, BE382707, BF316838, BE262654, AI344021, AW451305, AW291138, AW301011, BE784787, BG026465, AW957567, BG253034, AI807860, AI970736, N99472, AA378218, H23090, W31086, BE185755, AK025935, AF258545. HDPPA04 224 904765 1-2392 15-2406 AU135908, AI990290, AW961323, AI798762, AA044757, AW105205, AW197379, AU156359, AA039608, AA247117, AW889458, AA303575, AA036918, AA247128, AI214428, AW449368, AA044631, AI762460, AL162253.17, AK001872.1, AF344424.1, AF329193.1. HE2OA95 230 637595 1-1657 15-1671 AI620217, AL528146, AI056665, AW964795, AI744518, AI038199, AA236476, AA747220, AI368718, AI888960, N47941, AA234584, H19150, AI360711, BF445507, BF475619, AI682413, AI468582, H05762, AA297549, R52959, N46864, T27100, AA736703, AA579494, H18483, BE855666, Z40989, AI474169, R41378, AW973144, BF982162, N49148, AL157430, AF179274, AR052523, AB041565, AB017270, AR052522, AB004064. HKABZ65 232 862030 1-1175 15-1189 AA715814, AA503019, AV762033, BE155099, AV734997, BF917346, AW338860, AC011666.28, AF242518.1, AF109907.1, AC004867.5, AC020917.4, AC004166.12, AL356915.19, AC005071.2, AC004878.2, AC005052.2, AC005081.3, AC002549.1, AL590763.1, AC020663.1, AC006064.9, AC008745.6, AC004858.2, AC022405.5, AC007666.12, AC008750.7, AL451144.5, AP001716.1, AC009131.6, AC004656.1, AL109825.23, AL355312.24, AL035086.12, AC010605.4, AC004067.1, AC004477.1, AC008736.6, AL109915.10, AC006023.2, AL033529.25, AC007637.9, AL139317.5, AL031311.1, AL049776.3, AC004971.3, AC009220.10, AL080243.21, AC005015.2, AC004686.1, AL022318.2, AC002310.1, AC009123.6, Z93015.9, AC021999.4, AL355353.23, AL050318.13, AL161756.6, AC011464.5, AL132712.4, AL359513.12, AC007546.5, AP001695.1, AL035683.9, AC018711.4, D87675.1, AL133444.4, AL139100.9, AF030453.1, AC006077.1, AC008895.7, AP001713.1, Z84487.2, AL357153.4, AL163636.6, AL359382.23, AC004770.1, AP001972.4, AC004675.1, AL355392.7, AC020906.6, AL138784.30, AC020754.4, AL162426.20, AC002288.1, AC009068.10, AC008101.15, AC008623.4, AC008891.7, Z98884.11, AL136137.15, AC011247.10, AL133163.2, AP001727.1, AC005098.2, AC004659.1, AC005670.1, AL139022.4, AC009812.17, AF088219.1, AL035404.20, AL139801.17, AF228703.1, AC002492.1, AC006084.1, AL353594.13, AC005077.5, AL160271.19, AP001724.1, AC008537.5, AC024561.4, AL139353.3, AC004491.1, AC008626.5, AL391987.15, AC010530.7, AP003352.2, AC009267.15, AL122013.5, AP000008.1, AC087071.2, AC009314.4, AC020913.6, AL078463.11, AL096700.14, AC002369.1, AC010102.3, AP003357.2, AL031123.14, Z95331.2, AL513008.14, AL118501.22, AP001435.2, AC005200.1, AJ400877.1, AC011469.6, AC016772.8, AC005089.2, AC005088.2, AF312912.1, AL022316.2, AL080317.11, AP001693.1, AP000553.1, AL390294.19, AC006345.4, AC091394.2, AL359813.23, AC007283.3, AL353807.18, AL109921.21, AC074121.16, Z98742.5, AC007383.4, AF243527.1, AC027130.5, AC010504.7, AL035462.21, AC010650.8, AC005180.2, AF334404.1, AL139187.19, AC005037.2, AL021391.2.

Table 4

Table 4 provides a key to the tissue/cell source identifier code disclosed in Table 1B.2, column 5. Column 1 of Table 4 provides the tissue/cell source identifier code disclosed in Table 1B.2, column 5. Columns 2-5 provide a description of the tissue or cell source. Note that “Description” and “Tissue” sources (i.e. columns 2 and 3) having the prefix “a_” indicates organs, tissues, or cells derived from “adult” sources. Codes corresponding to diseased tissues are indicated in column 6 with the word “disease.” The use of the word “disease” in column 6 is non-limiting. The tissue or cell source may be specific (e.g. a neoplasm), or may be disease-associated (e.g., a tissue sample from a normal portion of a diseased organ). Furthermore, tissues and/or cells lacking the “disease” designation may still be derived from sources directly or indirectly involved in a disease state or disorder, and therefore may have a further utility in that disease state or disorder. In numerous cases where the tissue/cell source is a library, column 7 identifies the vector used to generate the library.

TABLE 4 Code Description Tissue Organ Cell Line Disease Vector AR022 a_Heart a_Heart AR023 a_Liver a_Liver AR024 a_mammary gland a_mammary gland AR025 a_Prostate a_Prostate AR026 a_small intestine a_small intestine AR027 a_Stomach a_Stomach AR028 Blood B cells Blood B cells AR029 Blood B cells activated Blood B cells activated AR030 Blood B cells resting Blood B cells resting AR031 Blood T cells activated Blood T cells activated AR032 Blood T cells resting Blood T cells resting AR033 brain brain AR034 breast breast AR035 breast cancer breast cancer AR036 Cell Line CAOV3 Cell Line CAOV3 AR037 cell line PA-1 cell line PA-1 AR038 cell line transformed cell line transformed AR039 colon colon AR040 colon (9808co65R) colon (9808co65R) AR041 colon (9809co15) colon (9809co15) AR042 colon cancer colon cancer AR043 colon cancer (9808co64R) colon cancer (9808co64R) AR044 colon cancer 9809co14 colon cancer 9809co14 AR045 corn clone 5 corn clone 5 AR046 corn clone 6 corn clone 6 AR047 corn clone 2 corn clone 2 AR048 corn clone 3 corn clone 3 AR049 Corn Clone 4 Corn Clone 4 AR050 Donor II B Cells 24 hrs Donor II B Cells 24 hrs AR051 Donor II B Cells 72 hrs Donor II B Cells 72 hrs AR052 Donor II B-Cells 24 hrs. Donor II B-Cells 24 hrs. AR053 Donor II B-Cells 72 hrs Donor II B-Cells 72 hrs AR054 Donor II Resting B Cells Donor II Resting B Cells AR055 Heart Heart AR056 Human Lung (CLONTECH ™) Human Lung (CLONTECH ™) AR057 Human Mammary Human Mammary (CLONTECH ™) (CLONTECH ™) AR058 Human Thymus (CLONTECH ™) Human Thymus (CLONTECH ™) AR059 Jurkat (unstimulated) Jurkat (unstimulated) AR060 Kidney Kidney AR061 Liver Liver AR062 Liver (CLONTECH ™) Liver (CLONTECH ™) AR063 Lymphocytes chronic Lymphocytes chronic lymphocytic leukaemia lymphocytic leukaemia AR064 Lymphocytes diffuse large B cell Lymphocytes diffuse large lymphoma B cell lymphoma AR065 Lymphocytes follicular Lymphocytes follicular lymphoma lymphoma AR066 normal breast normal breast AR067 Normal Ovarian (4004901) Normal Ovarian (4004901) AR068 Normal Ovary 9508G045 Normal Ovary 9508G045 AR069 Normal Ovary 9701G208 Normal Ovary 9701G208 AR070 Normal Ovary 9806G005 Normal Ovary 9806G005 AR071 Ovarian Cancer Ovarian Cancer AR072 Ovarian Cancer (9702G001) Ovarian Cancer (9702G001) AR073 Ovarian Cancer (9707G029) Ovarian Cancer (9707G029) AR074 Ovarian Cancer (9804G011) Ovarian Cancer (9804G011) AR075 Ovarian Cancer (9806G019) Ovarian Cancer (9806G019) AR076 Ovarian Cancer (9807G017) Ovarian Cancer (9807G017) AR077 Ovarian Cancer (9809G001) Ovarian Cancer (9809G001) AR078 ovarian cancer 15799 ovarian cancer 15799 AR079 Ovarian Cancer 17717AID Ovarian Cancer 17717AID AR080 Ovarian Cancer 4004664B1 Ovarian Cancer 4004664B1 AR081 Ovarian Cancer 4005315A1 Ovarian Cancer 4005315A1 AR082 ovarian cancer 94127303 ovarian cancer 94127303 AR083 Ovarian Cancer 96069304 Ovarian Cancer 96069304 AR084 Ovarian Cancer 9707G029 Ovarian Cancer 9707G029 AR085 Ovarian Cancer 9807G045 Ovarian Cancer 9807G045 AR086 ovarian cancer 9809G001 ovarian cancer 9809G001 AR087 Ovarian Cancer 9905C032RC Ovarian Cancer 9905C032RC AR088 Ovarian cancer 9907 C00 3rd Ovarian cancer 9907 C00 3rd AR089 Prostate Prostate AR090 Prostate (CLONTECH ™) Prostate (CLONTECH ™) AR091 prostate cancer prostate cancer AR092 prostate cancer #15176 prostate cancer #15176 AR093 prostate cancer #15509 prostate cancer #15509 AR094 prostate cancer #15673 prostate cancer #15673 AR095 Small Intestine (CLONTECH ™) Small Intestine (CLONTECH ™) AR096 Spleen Spleen AR097 Thymus T cells activated Thymus T cells activated AR098 Thymus T cells resting Thymus T cells resting AR099 Tonsil Tonsil AR100 Tonsil geminal center centroblast Tonsil geminal center centroblast AR101 Tonsil germinal center B cell Tonsil germinal center B cell AR102 Tonsil lymph node Tonsil lymph node AR103 Tonsil memory B cell Tonsil memory B cell AR104 Whole Brain Whole Brain AR105 Xenograft ES-2 Xenograft ES-2 AR106 Xenograft SW626 Xenograft SW626 AR119 001: IL-2 001: IL-2 AR120 001: IL-2.1 001: IL-2.1 AR121 001: IL-2_b 001: IL-2_b AR124 002: Monocytes untreated (1 hr) 002: Monocytes untreated (1 hr) AR125 002: Monocytes untreated (5 hrs) 002: Monocytes untreated (5 hrs) AR126 002: Control.1C 002: Control.1C AR127 002: IL2.1C 002: IL2.1C AR130 003: Placebo-treated Rat 003: Placebo-treated Rat Lacrimal Gland Lacrimal Gland AR131 003: Placebo-treated Rat 003: Placebo-treated Rat Submandibular Gland Submandibular Gland AR135 004: Monocytes untreated (5 hrs) 004: Monocytes untreated (5 hrs) AR136 004: Monocytes untreated 1 hr 004: Monocytes untreated 1 hr AR139 005: Placebo (48 hrs) 005: Placebo (48 hrs) AR140 006: pC4 (24 hrs) 006: pC4 (24 hrs) AR141 006: pC4 (48 hrs) 006: pC4 (48 hrs) AR152 007: PHA(1 hr) 007: PHA(1 hr) AR153 007: PHA(6 HRS) 007: PHA(6 HRS) AR154 007: PMA(6 hrs) 007: PMA(6 hrs) AR155 008: 1449_#2 008: 1449_#2 AR161 01: A - max 24 01: A - max 24 AR162 01: A - max 26 01: A - max 26 AR163 01: A - max 30 01: A - max 30 AR164 01: B - max 24 01: B - max 24 AR165 01: B - max 26 01: B - max 26 AR166 01: B - max 30 01: B - max 30 AR167 1449 Sample 1449 Sample AR168 3T3P10 1.0 uM insulin 3T3P10 1.0 uM insulin AR169 3T3P10 10 nM Insulin 3T3P10 10 nM Insulin AR170 3T3P10 10 uM insulin 3T3P10 10 uM insulin AR171 3T3P10 No Insulin 3T3P10 No Insulin AR172 3T3P4 3T3P4 AR173 Adipose (41892) Adipose (41892) AR174 Adipose Diabetic (41611) Adipose Diabetic (41611) AR175 Adipose Diabetic (41661) Adipose Diabetic (41661) AR176 Adipose Diabetic (41689) Adipose Diabetic (41689) AR177 Adipose Diabetic (41706) Adipose Diabetic (41706) AR178 Adipose Diabetic (42352) Adipose Diabetic (42352) AR179 Adipose Diabetic (42366) Adipose Diabetic (42366) AR180 Adipose Diabetic (42452) Adipose Diabetic (42452) AR181 Adipose Diabetic (42491) Adipose Diabetic (42491) AR182 Adipose Normal (41843) Adipose Normal (41843) AR183 Adipose Normal (41893) Adipose Normal (41893) AR184 Adipose Normal (42452) Adipose Normal (42452) AR185 Adrenal Gland Adrenal Gland AR186 Adrenal Gland + Whole Brain Adrenal Gland + Whole Brain AR187 B7(1 hr) + (inverted) B7(1 hr) + (inverted) AR188 Breast (18275A2B) Breast (18275A2B) AR189 Breast (4004199) Breast (4004199) AR190 Breast (4004399) Breast (4004399) AR191 Breast (4004943B7) Breast (4004943B7) AR192 Breast (4005570B1) Breast (4005570B1) AR193 Breast Cancer (4004127A30) Breast Cancer (4004127A30) AR194 Breast Cancer (400443A21) Breast Cancer (400443A21) AR195 Breast Cancer (4004643A2) Breast Cancer (4004643A2) AR196 Breast Cancer (4004710A7) Breast Cancer (4004710A7) AR197 Breast Cancer (4004943A21) Breast Cancer (4004943A21) AR198 Breast Cancer (400553A2) Breast Cancer (400553A2) AR199 Breast Cancer (9805C046R) Breast Cancer (9805C046R) AR200 Breast Cancer (9806C012R) Breast Cancer (9806C012R) AR201 Breast Cancer (ODQ 45913) Breast Cancer (ODQ 45913) AR202 Breast Cancer (ODQ45913) Breast Cancer (ODQ45913) AR203 Breast Cancer (ODQ4591B) Breast Cancer (ODQ4591B) AR204 Colon Cancer (15663) Colon Cancer (15663) AR205 Colon Cancer (4005144A4) Colon Cancer (4005144A4) AR206 Colon Cancer (4005413A4) Colon Cancer (4005413A4) AR207 Colon Cancer (4005570B1) Colon Cancer (4005570B1) AR208 Control RNA #1 Control RNA #1 AR209 Control RNA #2 Control RNA #2 AR210 Cultured Preadipocyte (blue) Cultured Preadipocyte (blue) AR211 Cultured Preadipocyte (Red) Cultured Preadipocyte (Red) AR212 Donor II B-Cells 24 hrs Donor II B-Cells 24 hrs AR213 Donor II Resting B-Cells Donor II Resting B-Cells AR214 H114EP12 10 nM Insulin H114EP12 10 nM Insulin AR215 H114EP12 (10 nM insulin) H114EP12 (10 nM insulin) AR216 H114EP12 (2.6 ug/ul) H114EP12 (2.6 ug/ul) AR217 H114EP12 (3.6 ug/ul) H114EP12 (3.6 ug/ul) AR218 HUVEC #1 HUVEC #1 AR219 HUVEC #2 HUVEC #2 AR221 L6 undiff. L6 undiff. AR222 L6 Undifferentiated L6 Undifferentiated AR223 L6P8 + 10 nM Insulin L6P8 + 10 nM Insulin AR224 L6P8 + HS L6P8 + HS AR225 L6P8 10 nM Insulin L6P8 10 nM Insulin AR226 Liver (00-06-A007B) Liver (00-06-A007B) AR227 Liver (96-02-A075) Liver (96-02-A075) AR228 Liver (96-03-A144) Liver (96-03-A144) AR229 Liver (96-04-A138) Liver (96-04-A138) AR230 Liver (97-10-A074B) Liver (97-10-A074B) AR231 Liver (98-09-A242A) Liver (98-09-A242A) AR232 Liver Diabetic (1042) Liver Diabetic (1042) AR233 Liver Diabetic (41616) Liver Diabetic (41616) AR234 Liver Diabetic (41955) Liver Diabetic (41955) AR235 Liver Diabetic (42352R) Liver Diabetic (42352R) AR236 Liver Diabetic (42366) Liver Diabetic (42366) AR237 Liver Diabetic (42483) Liver Diabetic (42483) AR238 Liver Diabetic (42491) Liver Diabetic (42491) AR239 Liver Diabetic (99-09-A281A) Liver Diabetic (99-09- A281A) AR240 Lung Lung AR241 Lung (27270) Lung (27270) AR242 Lung (2727Q) Lung (2727Q) AR243 Lung Cancer (4005116A1) Lung Cancer (4005116A1) AR244 Lung Cancer (4005121A5) Lung Cancer (4005121A5) AR245 Lung Cancer (4005121A5)) Lung Cancer (4005121A5)) AR246 Lung Cancer (4005340A4) Lung Cancer (4005340A4) AR247 Mammary Gland Mammary Gland AR248 Monocyte (CT) Monocyte (CT) AR249 Monocyte (OCT) Monocyte (OCT) AR250 Monocytes (CT) Monocytes (CT) AR251 Monocytes (INFG 18 hr) Monocytes (INFG 18 hr) AR252 Monocytes (INFG 18 hr) Monocytes (INFG 18 hr) AR253 Monocytes (INFG 8-11) Monocytes (INFG 8-11) AR254 Monocytes (O CT) Monocytes (O CT) AR255 Muscle (91-01-A105) Muscle (91-01-A105) AR256 Muscle (92-04-A059) Muscle (92-04-A059) AR257 Muscle (97-11-A056d) Muscle (97-11-A056d) AR258 Muscle (99-06-A210A) Muscle (99-06-A210A) AR259 Muscle (99-07-A203B) Muscle (99-07-A203B) AR260 Muscle (99-7-A203B) Muscle (99-7-A203B) AR261 Muscle Diabetic (42352R) Muscle Diabetic (42352R) AR262 Muscle Diabetic (42366) Muscle Diabetic (42366) AR263 NK-19 Control NK-19 Control AR264 NK-19 IL Treated 72 hrs NK-19 IL Treated 72 hrs AR265 NK-19 UK Treated 72 hrs. NK-19 UK Treated 72 hrs. AR266 Omentum Normal (94-08-B009) Omentum Normal (94-08- B009) AR267 Omentum Normal (97-01- Omentum Normal (97-01- A039A) A039A) AR268 Omentum Normal (97-04- Omentum Normal (97-04- A114C) A114C) AR269 Omentum Normal (97-06- Omentum Normal (97-06- A117C) A117C) AR270 Omentum Normal (97-09-B004C) Omentum Normal (97-09- B004C) AR271 Ovarian Cancer (17717AID) Ovarian Cancer (17717AID) AR272 Ovarian Cancer (9905C023RC) Ovarian Cancer (9905C023RC) AR273 Ovarian Cancer (9905C032RC) Ovarian Cancer (9905C032RC) AR274 Ovary (9508G045) Ovary (9508G045) AR275 Ovary (9701G208) Ovary (9701G208) AR276 Ovary 9806G005 Ovary 9806G005 AR277 Pancreas Pancreas AR278 Placebo Placebo AR279 rIL2 Control rIL2 Control AR280 RSS288L RSS288L AR281 RSS288LC RSS288LC AR282 Salivary Gland Salivary Gland AR283 Skeletal Muscle Skeletal Muscle AR284 Skeletal Muscle (91-01-A105) Skeletal Muscle (91-01- A105) AR285 Skeletal Muscle (42180) Skeletal Muscle (42180) AR286 Skeletal Muscle (42386) Skeletal Muscle (42386) AR287 Skeletal Muscle (42461) Skeletal Muscle (42461) AR288 Skeletal Muscle (91-01-A105) Skeletal Muscle (91-01- A105) AR289 Skeletal Muscle (92-04-A059) Skeletal Muscle (92-04- A059) AR290 Skeletal Muscle (96-08-A171) Skeletal Muscle (96-08- A171) AR291 Skeletal Muscle (97-07-A190A) Skeletal Muscle (97-07- A190A) AR292 Skeletal Muscle Diabetic (42352) Skeletal Muscle Diabetic (42352) AR293 Skeletal Muscle Diabetic (42366) Skeletal Muscle Diabetic (42366) AR294 Skeletal Muscle Diabetic (42395) Skeletal Muscle Diabetic (42395) AR295 Skeletal Muscle Diabetic (42483) Skeletal Muscle Diabetic (42483) AR296 Skeletal Muscle Diabetic (42491) Skeletal Muscle Diabetic (42491) AR297 Skeletal Muscle Diabetic 42352 Skeletal Muscle Diabetic 42352 AR298 Skeletal Musle (42461) Skeletal Musle (42461) AR299 Small Intestine Small Intestine AR300 Stomach Stomach AR301 T-Cell + HDPBQ71.fc 1449 T-Cell + HDPBQ71.fc 16 hrs 1449 16 hrs AR302 T-Cell + HDPBQ71.fc 1449 6 hrs T-Cell + HDPBQ71.fc 1449 6 hrs AR303 T-Cell + IL2 16 hrs T-Cell + IL2 16 hrs AR304 T-Cell + IL2 6 hrs T-Cell + IL2 6 hrs AR306 T-Cell Untreated 16 hrs T-Cell Untreated 16 hrs AR307 T-Cell Untreated 6 hrs T-Cell Untreated 6 hrs AR308 T-Cells 24 hours T-Cells 24 hours AR309 T-Cells 24 hrs T-Cells 24 hrs AR310 T-Cells 24 hrs. T-Cells 24 hrs. AR311 T-Cells 24 hrs T-Cells 24 hrs AR312 T-Cells 4 days T-Cells 4 days AR313 Thymus Thymus AR314 TRE TRE AR315 TREC TREC AR316 Virtual Mixture Virtual Mixture AR317 B lymphocyte, B lymphocyte, AR318 (non-T; non-B) (non-T; non-B) AR326 001-293 RNA (Vector Control) 001-293 RNA (Vector Control) AR327 001: Control 001: Control AR328 001: Control.1 001: Control.1 AR355 Acute Lymphocyte Leukemia Acute Lymphocyte Leukemia AR356 AML Patient #11 AML Patient #11 AR357 AML Patient #2 AML Patient #2 AR358 AML Patient #2 SGAH AML Patient #2 SGAH AR359 AML Patient#2 AML Patient#2 AR360 Aorta Aorta AR361 B Cell B Cell AR362 B lymphoblast B lymphoblast AR363 B lymphocyte B lymphocyte AR364 B lymphocytes B lymphocytes AR365 B-cell B-cell AR366 B-Cells B-Cells AR367 B-Lymphoblast B-Lymphoblast AR368 B-Lymphocytes B-Lymphocytes AR369 Bladder Bladder AR370 Bone Marrow Bone Marrow AR371 Bronchial Epithelial Cell Bronchial Epithelial Cell AR372 Bronchial Epithelial Cells Bronchial Epithelial Cells AR373 Caco-2A Caco-2A AR374 Caco-2B Caco-2B AR375 Caco-2C Caco-2C AR376 Cardiac #1 Cardiac #1 AR377 Cardiac #2 Cardiac #2 AR378 Chest Muscle Chest Muscle AR381 Dendritic Cell Dendritic Cell AR382 Dendritic cells Dendritic cells AR383 E. coli E. coli AR384 Epithelial Cells Epithelial Cells AR385 Esophagus Esophagus AR386 FPPS FPPS AR387 FPPSC FPPSC AR388 HepG2 Cell Line HepG2 Cell Line AR389 HepG2 Cell line Buffer 1 hr. HepG2 Cell line Buffer 1 hr. AR390 HepG2 Cell line Buffer 06 hr HepG2 Cell line Buffer 06 hr AR391 HepG2 Cell line Buffer 24 hr. HepG2 Cell line Buffer 24 hr. AR392 HepG2 Cell line Insulin 01 hr. HepG2 Cell line Insulin 01 hr. AR393 HepG2 Cell line Insulin 06 hr. HepG2 Cell line Insulin 06 hr. AR394 HepG2 Cell line Insulin 24 hr. HepG2 Cell line Insulin 24 hr. AR398 HMC-1 HMC-1 AR399 HMCS HMCS AR400 HMSC HMSC AR401 HUVEC #3 HUVEC #3 AR402 HUVEC #4 HUVEC #4 AR404 KIDNEY NORMAL KIDNEY NORMAL AR405 KIDNEY TUMOR KIDNEY TUMOR AR406 KIDNEY TUMOR AR407 Lymph Node Lymph Node AR408 Macrophage Macrophage AR409 Megakarioblast Megakarioblast AR410 Monocyte Monocyte AR411 Monocytes Monocytes AR412 Myocardium Myocardium AR413 Myocardium #3 Myocardium #3 AR414 Myocardium #4 Myocardium #4 AR415 Myocardium #5 Myocardium #5 AR416 NK NK AR417 NK cell NK cell AR418 NK cells NK cells AR419 NKYa NKYa AR420 NKYa019 NKYa019 AR421 Ovary Ovary AR422 Patient #11 Patient #11 AR423 Peripheral blood Peripheral blood AR424 Primary Adipocytes Primary Adipocytes AR425 Promyeloblast Promyeloblast AR427 RSSWT RSSWT AR428 RSSWTC RSSWTC AR429 SW 480(G1) SW 480(G1) AR430 SW 480(G2) SW 480(G2) AR431 SW 480(G3) SW 480(G3) AR432 SW 480(G4) SW 480(G4) AR433 SW 480(G5) SW 480(G5) AR434 T Lymphoblast T Lymphoblast AR435 T Lymphocyte T Lymphocyte AR436 T-Cell T-Cell AR438 T-Cell, T-Cell, AR439 T-Cells T-Cells AR440 T-lymphoblast T-lymphoblast AR441 Th 1 Th 1 AR442 Th 2 Th 2 AR443 Th1 Th1 AR444 Th2 Th2 H0002 Human Adult Heart Human Adult Heart Heart UNI-ZAP ™ XR H0003 Human Adult Liver Human Adult Liver Liver UNI-ZAP ™ XR H0004 Human Adult Spleen Human Adult Spleen Spleen UNI-ZAP ™ XR H0006 Human Frontal Lobe of Brain UNI-ZAP ™ XR H0007 Human Cerebellum Human Cerebellum Brain UNI-ZAP ™ XR H0008 Whole 6 Week Old Embryo UNI-ZAP ™ XR H0009 Human Fetal Brain UNI-ZAP ™ XR H0009 Human Fetal Brain Human Fetal Brain Brain UNI-ZAP ™ XR H0010 Human Fetal Hepatic Human Fetal Liver Liver UNI-ZAP ™ XR H0011 Human Fetal Kidney Human Fetal Kidney Kidney UNI-ZAP ™ XR H0012 Human Fetal Kidney Human Fetal Kidney Kidney UNI-ZAP ™ XR H0013 Human 8 Week Whole Embryo Human 8 Week Old Embryo UNI-ZAP ™ Embryo XR H0014 Human Gall Bladder Human Gall Bladder Gall Bladder UNI-ZAP ™ XR H0015 Human Gall Bladder, fraction II Human Gall Bladder Gall Bladder UNI-ZAP ™ XR H0016 Human Greater Omentum Human Greater Omentum peritoneum UNI-ZAP ™ XR H0017 Human Greater Omentum Human Greater Omentum peritoneum UNI-ZAP ™ XR H0018 Human Greater Omentum, fII Human Greater Omentum peritoneum UNI-ZAP ™ remake XR H0019 Human Fetal Heart Human Fetal Heart Heart pBLUESCRIPT ™ H0020 Human Hippocampus Human Hippocampus Brain UNI-ZAP ™ XR H0021 Human Infant Adrenal Gland Human Infant Adrenal Adrenal UNI-ZAP ™ Gland gland XR H0022 Jurkat Cells Jurkat T-Cell Line LAMBDA ZAP ™ II H0023 Human Fetal Lung UNI-ZAP ™ XR H0024 Human Fetal Lung III Human Fetal Lung Lung UNI-ZAP ™ XR H0025 Human Adult Lymph Node Human Adult Lymph Node Lymph Node LAMBDA ZAP ™ II H0026 Namalwa Cells Namalwa B-Cell Line, LAMBDA EBV immortalized ZAP ™ II H0028 Human Old Ovary Human Old Ovary Ovary pBLUESCRIPT ™ H0029 Human Pancreas Human Pancreas Pancreas UNI-ZAP ™ XR H0030 Human Placenta UNI-ZAP ™ XR H0031 Human Placenta Human Placenta Placenta UNI-ZAP ™ XR H0032 Human Prostate Human Prostate Prostate UNI-ZAP ™ XR H0033 Human Pituitary Human Pituitary UNI-ZAP ™ XR H0034 Human Parathyroid Tumor Human Parathyroid Tumor Parathyroid disease UNI-ZAP ™ XR H0035 Human Salivary Gland Human Salivary Gland Salivary UNI-ZAP ™ gland XR H0036 Human Adult Small Intestine Human Adult Small Small Int. UNI-ZAP ™ Intestine XR H0037 Human Adult Small Intestine Human Adult Small Small Int. pBLUESCRIPT ™ Intestine H0038 Human Testes Human Testes Testis UNI-ZAP ™ XR H0039 Human Pancreas Tumor Human Pancreas Tumor Pancreas disease UNI-ZAP ™ XR H0040 Human Testes Tumor Human Testes Tumor Testis disease UNI-ZAP ™ XR H0041 Human Fetal Bone Human Fetal Bone Bone UNI-ZAP ™ XR H0042 Human Adult Pulmonary Human Adult Pulmonary Lung UNI-ZAP ™ XR H0044 Human Cornea Human Cornea eye UNI-ZAP ™ XR H0045 Human Esophagus, Cancer Human Esophagus, cancer Esophagus disease UNI-ZAP ™ XR H0046 Human Endometrial Tumor Human Endometrial Tumor Uterus disease UNI-ZAP ™ XR H0047 Human Fetal Liver Human Fetal Liver Liver UNI-ZAP ™ XR H0048 Human Pineal Gland Human Pineal Gland UNI-ZAP ™ XR H0049 Human Fetal Kidney Human Fetal Kidney Kidney UNI-ZAP ™ XR H0050 Human Fetal Heart Human Fetal Heart Heart UNI-ZAP ™ XR H0051 Human Hippocampus Human Hippocampus Brain UNI-ZAP ™ XR H0052 Human Cerebellum Human Cerebellum Brain UNI-ZAP ™ XR H0053 Human Adult Kidney Human Adult Kidney Kidney UNI-ZAP ™ XR H0056 Human Umbilical Vein, Endo. Human Umbilical Vein Umbilical UNI-ZAP ™ remake Endothelial Cells vein XR H0057 Human Fetal Spleen UNI-ZAP ™ XR H0058 Human Thymus Tumor Human Thymus Tumor Thymus disease LAMBDA ZAP ™ II H0059 Human Uterine Cancer Human Uterine Cancer Uterus disease LAMBDA ZAP ™ II H0060 Human Macrophage Human Macrophage Blood Cell Line pBLUESCRIPT ™ H0061 Human Macrophage Human Macrophage Blood Cell Line pBLUESCRIPT ™ H0062 Human Thymus Human Thymus Thymus UNI-ZAP ™ XR H0063 Human Thymus Human Thymus Thymus UNI-ZAP ™ XR H0064 Human Right Hemisphere of Human Brain, right Brain UNI-ZAP ™ Brain hemisphere XR H0065 Human Esophagus, Normal Human Esophagus, normal Esophagus UNI-ZAP ™ XR H0067 Human left hemisphere, adult Human Left Hemisphere, Brain LAMBDA Adult ZAP ™ II H0068 Human Skin Tumor Human Skin Tumor Skin disease UNI-ZAP ™ XR H0069 Human Activated T-Cells Activated T-Cells Blood Cell Line UNI-ZAP ™ XR H0070 Human Pancreas Human Pancreas Pancreas UNI-ZAP ™ XR H0071 Human Infant Adrenal Gland Human Infant Adrenal Adrenal UNI-ZAP ™ Gland gland XR H0073 Human Leiomyeloid Carcinoma Human Leiomyeloid Muscle disease UNI-ZAP ™ Carcinoma XR H0074 Human Platelets Human Platelets Blood Cell Line UNI-ZAP ™ XR H0075 Human Activated T-Cells (II) Activated T-Cells Blood Cell Line UNI-ZAP ™ XR H0076 Human Membrane Bound Human Membrane Bound Blood Cell Line UNI-ZAP ™ Polysomes Polysomes XR H0077 Human Thymus Tumor Human Thymus Tumor Thymus disease LAMBDA ZAP ™ II H0078 Human Lung Cancer Human Lung Cancer Lung disease LAMBDA ZAP ™ II H0079 Human Whole 7 Week Old Human Whole 7 Week Old Embryo UNI-ZAP ™ Embryo (II) Embryo XR H0080 Human Whole 6 Week Old Human Whole Six Week Embryo LAMBDA Embryo (II) Old Embryo ZAP ™ II H0081 Human Fetal Epithelium (Skin) Human Fetal Skin Skin UNI-ZAP ™ XR H0082 Human Fetal Muscle Human Fetal Muscle Sk Muscle UNI-ZAP ™ XR H0083 HUMAN JURKAT Jurkat Cells UNI-ZAP ™ MEMBRANE BOUND XR POLYSOMES H0085 Human Colon Human Colon LAMBDA ZAP ™ II H0086 Human epithelioid sarcoma Epithelioid Sarcoma, Sk Muscle disease UNI-ZAP ™ muscle XR H0087 Human Thymus Human Thymus pBLUESCRIPT ™ H0090 Human T-Cell Lymphoma T-Cell Lymphoma T-Cell disease UNI-ZAP ™ XR H0092 Human Pancreas Tumor Human Pancreas Tumor Pancreas disease UNI-ZAP ™ XR H0093 Human Greater Omentum Tumor Human Greater Omentum peritoneum disease UNI-ZAP ™ XR H0095 Human Greater Omentum, RNA Human Greater Omentum peritoneum UNI-ZAP ™ Remake XR H0096 Human Parotid Cancer Human Parotid Cancer Parotid disease LAMBDA ZAP ™ II H0097 Human Adult Heart, subtracted Human Adult Heart Heart pBLUESCRIPT ™ H0098 Human Adult Liver, subtracted Human Adult Liver Liver UNI-ZAP ™ XR H0099 Human Lung Cancer, subtracted Human Lung Cancer Lung pBLUESCRIPT ™ H0100 Human Whole Six Week Old Human Whole Six Week Embryo UNI-ZAP ™ Embryo Old Embryo XR H0101 Human 7 Weeks Old Embryo, Human Whole 7 Week Old Embryo LAMBDA subtracted Embryo ZAP ™ II H0102 Human Whole 6 Week Old Human Whole Six Week Embryo pBLUESCRIPT ™ Embryo (II), subt Old Embryo H0103 Human Fetal Brain, subtracted Human Fetal Brain Brain UNI-ZAP ™ XR H0105 Human Fetal Heart, subtracted Human Fetal Heart Heart pBLUESCRIPT ™ H0106 Human Right Hemisphere of Human Brain, right Brain UNI-ZAP ™ Brain, subtrac hemisphere XR H0107 Human Infant Adrenal Gland, Human Infant Adrenal Adrenal pBLUESCRIPT ™ subtracted Gland gland H0108 Human Adult Lymph Node, Human Adult Lymph Node Lymph Node UNI-ZAP ™ subtracted XR H0109 Human Macrophage, subtracted Macrophage Blood Cell Line pBLUESCRIPT ™ H0110 Human Old Ovary, subtracted Human Old Ovary Ovary pBLUESCRIPT ™ H0111 Human Placenta, subtracted Human Placenta Placenta pBLUESCRIPT ™ H0112 Human Parathyroid Tumor, Human Parathyroid Tumor Parathyroid pBLUESCRIPT ™ subtracted H0113 Human skin Tumor, subtracted Human Skin Tumor Skin UNI-ZAP ™ XR H0116 Human Thymus Tumor, Human Thymus Tumor Thymus pBLUESCRIPT ™ subtracted H0117 Human Uterine Cancer, Human Uterine Cancer Uterus pBLUESCRIPT ™ subtracted H0118 Human Adult Kidney Human Adult Kidney Kidney UNI-ZAP ™ XR H0119 Human Pediatric Kidney Human Pediatric Kidney Kidney UNI-ZAP ™ XR H0120 Human Adult Spleen, subtracted Human Adult Spleen Spleen UNI-ZAP ™ XR H0121 Human Cornea, subtracted Human Cornea eye UNI-ZAP ™ XR H0122 Human Adult Skeletal Muscle Human Skeletal Muscle Sk Muscle UNI-ZAP ™ XR H0123 Human Fetal Dura Mater Human Fetal Dura Mater Brain UNI-ZAP ™ XR H0124 Human Rhabdomyosarcoma Human Sk Muscle disease UNI-ZAP ™ Rhabdomyosarcoma XR H0125 Cem cells cyclohexamide treated Cyclohexamide Treated Blood Cell Line UNI-ZAP ™ Cem, Jurkat, Raji, and Supt XR H0128 Jurkat cells, thiouridine activated Jurkat Cells UNI-ZAP ™ XR H0129 Jurkat cells, thiouridine activated, Jurkat Cells UNI-ZAP ™ fract II XR H0130 LNCAP untreated LNCAP Cell Line Prostate Cell Line UNI-ZAP ™ XR H0131 LNCAP + o.3 nM R1881 LNCAP Cell Line Prostate Cell Line UNI-ZAP ™ XR H0132 LNCAP + 30 nM R1881 LNCAP Cell Line Prostate Cell Line UNI-ZAP ™ XR H0134 Raji Cells, cyclohexamide treated Cyclohexamide Treated Blood Cell Line UNI-ZAP ™ Cem, Jurkat, Raji, and Supt XR H0135 Human Synovial Sarcoma Human Synovial Sarcoma Synovium UNI-ZAP ™ XR H0136 Supt Cells, cyclohexamide treated Cyclohexamide Treated Blood Cell Line UNI-ZAP ™ Cem, Jurkat, Raji, and Supt XR H0138 Activated T-Cells, 0 hrs. Activated T-Cells Blood Cell Line UNI-ZAP ™ XR H0139 Activated T-Cells, 4 hrs. Activated T-Cells Blood Cell Line UNI-ZAP ™ XR H0140 Activated T-Cells, 8 hrs. Activated T-Cells Blood Cell Line UNI-ZAP ™ XR H0141 Activated T-Cells, 12 hrs. Activated T-Cells Blood Cell Line UNI-ZAP ™ XR H0142 MCF7 Cell Line MCF7 Cell line Breast Cell Line UNI-ZAP ™ XR H0144 Nine Week Old Early Stage 9 Wk Old Early Stage Embryo UNI-ZAP ™ Human Human XR H0147 Human Adult Liver Human Adult Liver Liver UNI-ZAP ™ XR H0149 7 Week Old Early Stage Human, Human Whole 7 Week Old Embryo UNI-ZAP ™ subtracted Embryo XR H0150 Human Epididymus Epididymis Testis UNI-ZAP ™ XR H0151 Early Stage Human Liver Human Fetal Liver Liver UNI-ZAP ™ XR H0152 Early Stage Human Liver, fract Human Fetal Liver Liver UNI-ZAP ™ (II) XR H0154 Human Fibrosarcoma Human Skin Fibrosarcoma Skin disease UNI-ZAP ™ XR H0155 Human Thymus, subtracted Human Thymus Tumor Thymus pBLUESCRIPT ™ H0156 Human Adrenal Gland Tumor Human Adrenal Gland Adrenal disease UNI-ZAP ™ Tumor Gland XR H0157 Activated T-Cells, 0 hrs, ligation 2 Activated T-Cells Blood Cell Line UNI-ZAP ™ XR H0158 Activated T-Cells, 4 hrs., ligation 2 Activated T-Cells Blood Cell Line UNI-ZAP ™ XR H0159 Activated T-Cells, 8 hrs., ligation 2 Activated T-Cells Blood Cell Line UNI-ZAP ™ XR H0160 Activated T-Cells, 12 hrs., Activated T-Cells Blood Cell Line UNI-ZAP ™ ligation 2 XR H0161 Activated T-Cells, 24 hrs., Activated T-Cells Blood Cell Line UNI-ZAP ™ ligation 2 XR H0163 Human Synovium Human Synovium Synovium UNI-ZAP ™ XR H0164 Human Trachea Tumor Human Trachea Tumor Trachea disease UNI-ZAP ™ XR H0165 Human Prostate Cancer, Stage B2 Human Prostate Cancer, Prostate disease UNI-ZAP ™ stage B2 XR H0166 Human Prostate Cancer, Stage B2 Human Prostate Cancer, Prostate disease UNI-ZAP ™ fraction stage B2 XR H0167 Activated T-Cells, 24 hrs. Activated T-Cells Blood Cell Line UNI-ZAP ™ XR H0168 Human Prostate Cancer, Stage C Human Prostate Cancer, Prostate disease UNI-ZAP ™ stage C XR H0169 Human Prostate Cancer, Stage C Human Prostate Cancer, Prostate disease UNI-ZAP ™ fraction stage C XR H0170 12 Week Old Early Stage Human Twelve Week Old Early Embryo UNI-ZAP ™ Stage Human XR H0171 12 Week Old Early Stage Human, Twelve Week Old Early Embryo UNI-ZAP ™ II Stage Human XR H0172 Human Fetal Brain, random Human Fetal Brain Brain LAMBDA primed ZAP ™ II H0173 Human Cardiomyopathy, RNA Human Cardiomyopathy Heart disease UNI-ZAP ™ remake XR H0175 H. Adult Spleen, ziplox pSport1 H0176 CAMA1Ee Cell Line CAMA1Ee Cell Line Breast Cell Line UNI-ZAP ™ XR H0177 CAMA1Ee Cell Line CAMA1Ee Cell Line Breast Cell Line UNI-ZAP ™ XR H0178 Human Fetal Brain Human Fetal Brain Brain UNI-ZAP ™ XR H0179 Human Neutrophil Human Neutrophil Blood Cell Line UNI-ZAP ™ XR H0180 Human Primary Breast Cancer Human Primary Breast Breast disease UNI-ZAP ™ Cancer XR H0181 Human Primary Breast Cancer Human Primary Breast Breast disease UNI-ZAP ™ Cancer XR H0182 Human Primary Breast Cancer Human Primary Breast Breast disease UNI-ZAP ™ Cancer XR H0183 Human Colon Cancer Human Colon Cancer Colon disease UNI-ZAP ™ XR H0184 Human Colon Cancer, Human Colon Cancer, Liver disease LAMBDA metasticized to live metasticized to liver ZAP ™ II H0185 Activated T-Cell labeled with 4- T-Cells Blood Cell Line LAMBDA thioluri ZAP ™ II H0186 Activated T-Cell T-Cells Blood Cell Line LAMBDA ZAP ™ II H0187 Resting T-Cell T-Cells Blood Cell Line LAMBDA ZAP ™ II H0188 Human Normal Breast Human Normal Breast Breast UNI-ZAP ™ XR H0189 Human Resting Macrophage Human Blood Cell Line UNI-ZAP ™ Macrophage/Monocytes XR H0190 Human Activated Macrophage Human Blood Cell Line UNI-ZAP ™ (LPS) Macrophage/Monocytes XR H0191 Human Activated Macrophage Human Blood Cell Line UNI-ZAP ™ (LPS), thiour Macrophage/Monocytes XR H0192 Cem Cells, cyclohexamide Cyclohexamide Treated Blood Cell Line UNI-ZAP ™ treated, subtra Cem, Jurkat, Raji, and Supt XR H0193 Cem Cells, cyclohexamide Cyclohexamide Treated Blood Cell Line UNI-ZAP ™ treated, differ Cem, Jurkat, Raji, and Supt XR H0194 Human Cerebellum, subtracted Human Cerebellum Brain pBLUESCRIPT ™ H0196 Human Cardiomyopathy, Human Cardiomyopathy Heart UNI-ZAP ™ subtracted XR H0197 Human Fetal Liver, subtracted Human Fetal Liver Liver UNI-ZAP ™ XR H0198 Human Fetal Liver, subtracted, Human Fetal Liver Liver UNI-ZAP ™ pos. clon XR H0199 Human Fetal Liver, subtracted, Human Fetal Liver Liver UNI-ZAP ™ neg clone XR H0200 Human Greater Omentum, fract II Human Greater Omentum peritoneum UNI-ZAP ™ remake, XR H0201 Human Hippocampus, subtracted Human Hippocampus Brain pBLUESCRIPT ™ H0202 Jurkat Cells, cyclohexamide Cyclohexamide Treated Blood Cell Line UNI-ZAP ™ treated, subtraction Cem, Jurkat, Raji, and Supt XR H0203 Jurkat Cells, cyclohexamide Cyclohexamide Treated Blood Cell Line UNI-ZAP ™ treated, dif Cem, Jurkat, Raji, and Supt XR H0204 Human Colon Cancer, subtracted Human Colon Cancer Colon pBLUESCRIPT ™ H0205 Human Colon Cancer, differential Human Colon Cancer Colon pBLUESCRIPT ™ H0207 LNCAP, differential expression LNCAP Cell Line Prostate Cell Line pBLUESCRIPT ™ H0208 Early Stage Human Lung, Human Fetal Lung Lung pBLUESCRIPT ™ subtracted H0209 Human Cerebellum, differentially Human Cerebellum Brain UNI-ZAP ™ expressed XR H0211 Human Prostate, differential Human Prostate Prostate pBLUESCRIPT ™ expression H0212 Human Prostate, subtracted Human Prostate Prostate pBLUESCRIPT ™ H0213 Human Pituitary, subtracted Human Pituitary UNI-ZAP ™ XR H0214 Raji cells, cyclohexamide treated, Cyclohexamide Treated Blood Cell Line pBLUESCRIPT ™ subtracted Cem, Jurkat, Raji, and Supt H0215 Raji cells, cyclohexamide treated, Cyclohexamide Treated Blood Cell Line pBLUESCRIPT ™ differentially expressed Cem, Jurkat, Raji, and Supt H0216 Supt cells, cyclohexamide treated, Cyclohexamide Treated Blood Cell Line pBLUESCRIPT ™ subtracted Cem, Jurkat, Raji, and Supt H0217 Supt cells, cyclohexamide treated, Cyclohexamide Treated Blood Cell Line pBLUESCRIPT ™ differentially expressed Cem, Jurkat, Raji, and Supt H0218 Activated T-Cells, 0 hrs, Activated T-Cells Blood Cell Line UNI-ZAP ™ subtracted XR H0219 Activated T-Cells, 0 hrs, Activated T-Cells Blood Cell Line UNI-ZAP ™ differentially expressed XR H0220 Activated T-Cells, 4 hrs, Activated T-Cells Blood Cell Line UNI-ZAP ™ subtracted XR H0221 Activated T-Cells, 4 hrs, Activated T-Cells Blood Cell Line UNI-ZAP ™ differentially expressed XR H0222 Activated T-Cells, 8 hrs, Activated T-Cells Blood Cell Line UNI-ZAP ™ subtracted XR H0223 Activated T-Cells, 8 hrs, Activated T-Cells Blood Cell Line UNI-ZAP ™ differentially expressed XR H0224 Activated T-Cells, 12 hrs, Activated T-Cells Blood Cell Line UNI-ZAP ™ subtracted XR H0225 Activated T-Cells, 12 hrs, Activated T-Cells Blood Cell Line UNI-ZAP ™ differentially expressed XR H0228 C7MCF7 cell line, estrogen C7MCF7 Cell Line, Breast Cell Line UNI-ZAP ™ treated estrogen treated XR H0229 Early Stage Human Brain, Early Stage Human Brain Brain LAMBDA random primed ZAP ™ II H0230 Human Cardiomyopathy, diff exp Human Cardiomyopathy Heart disease UNI-ZAP ™ XR H0231 Human Colon, subtraction Human Colon pBLUESCRIPT ™ H0232 Human Colon, differential Human Colon pBLUESCRIPT ™ expression H0233 Human Fetal Heart, Differential Human Fetal Heart Heart pBLUESCRIPT ™ (Adult-Specific) H0234 human colon cancer, metastatic to Human Colon Cancer, Liver pBLUESCRIPT ™ liver, differentially expressed metasticized to liver H0235 Human colon cancer, metaticized Human Colon Cancer, Liver pBLUESCRIPT ™ to liver, subtraction metasticized to liver H0238 Human Myometrium Leiomyoma Human Myometrium Uterus disease UNI-ZAP ™ Leiomyoma XR H0239 Human Kidney Tumor Human Kidney Tumor Kidney disease UNI-ZAP ™ XR H0240 C7MCF7 cell line, estrogen C7MCF7 Cell Line, Breast Cell Line UNI-ZAP ™ treated, Differential estrogen treated XR H0241 C7MCF7 cell line, estrogen C7MCF7 Cell Line, Breast Cell Line UNI-ZAP ™ treated, subtraction estrogen treated XR H0242 Human Fetal Heart, Differential Human Fetal Heart Heart pBLUESCRIPT ™ (Fetal-Specific) H0244 Human 8 Week Whole Embryo, Human 8 Week Old Embryo UNI-ZAP ™ subtracted Embryo XR H0246 Human Fetal Liver - Enzyme Human Fetal Liver Liver UNI-ZAP ™ subtraction XR H0247 Human Membrane Bound Human Membrane Bound Blood Cell Line UNI-ZAP ™ Polysomes - Enzyme Subtraction Polysomes XR H0249 HE7, subtracted by hybridization Human Whole 7 Week Old Embryo UNI-ZAP ™ with E7 cDNA Embryo XR H0250 Human Activated Monocytes Human Monocytes UNI-ZAP ™ XR H0251 Human Chondrosarcoma Human Chondrosarcoma Cartilage disease UNI-ZAP ™ XR H0252 Human Osteosarcoma Human Osteosarcoma Bone disease UNI-ZAP ™ XR H0253 Human adult testis, large inserts Human Adult Testis Testis UNI-ZAP ™ XR H0254 Breast Lymph node cDNA library Breast Lymph Node Lymph Node UNI-ZAP ™ XR H0255 breast lymph node CDNA library Breast Lymph Node Lymph Node LAMBDA ZAP ™ II H0256 HL-60, unstimulated Human HL-60 Cells, Blood Cell Line UNI-ZAP ™ unstimulated XR H0257 HL-60, PMA 4 H HL-60 Cells, PMA Blood Cell Line UNI-ZAP ™ stimulated 4 H XR H0261 H. cerebellum, Enzyme Human Cerebellum Brain UNI-ZAP ™ subtracted XR H0263 human colon cancer Human Colon Cancer Colon disease LAMBDA ZAP ™ II H0264 human tonsils Human Tonsil Tonsil UNI-ZAP ™ XR H0265 Activated T-Cell T-Cells Blood Cell Line UNI-ZAP ™ (12 hs)/Thiouridine labelledEco XR H0266 Human Microvascular HMEC Vein Cell Line LAMBDA Endothelial Cells, fract. A ZAP ™ II H0267 Human Microvascular HMEC Vein Cell Line LAMBDA Endothelial Cells, fract. B ZAP ™ II H0268 Human Umbilical Vein HUVE Cells Umbilical Cell Line LAMBDA Endothelial Cells, fract. A vein ZAP ™ II H0269 Human Umbilical Vein HUVE Cells Umbilical Cell Line LAMBDA Endothelial Cells, fract. B vein ZAP ™ II H0270 HPAS (human pancreas, Human Pancreas Pancreas UNI-ZAP ™ subtracted) XR H0271 Human Neutrophil, Activated Human Neutrophil- Blood Cell Line UNI-ZAP ™ Activated XR H0272 HUMAN TONSILS, FRACTION 2 Human Tonsil Tonsil UNI-ZAP ™ XR H0274 Human Adult Spleen, fractionII Human Adult Spleen Spleen UNI-ZAP ™ XR H0275 Human Infant Adrenal Gland, Human Infant Adrenal Adrenal pBLUESCRIPT ™ Subtracted Gland gland H0279 K562 cells K562 Cell line cell line Cell Line ZAP EXPRESS ™ H0280 K562 + PMA (36 hrs) K562 Cell line cell line Cell Line ZAP EXPRESS ™ H0281 Lymph node, abnorm. cell line Lymph Node, abnormal cell Lymph Node Cell Line ZAP (ATCC ™ #7225) line EXPRESS ™ H0282 HBGB''s differential Human Primary Breast Breast UNI-ZAP ™ consolidation Cancer XR H0284 Human OB MG63 control Human Osteoblastoma Bone Cell Line UNI-ZAP ™ fraction I MG63 cell line XR H0286 Human OB MG63 treated (10 nM Human Osteoblastoma Bone Cell Line UNI-ZAP ™ E2) fraction I MG63 cell line XR H0288 Human OB HOS control fraction I Human Osteoblastoma Bone Cell Line UNI-ZAP ™ HOS cell line XR H0290 Human OB HOS treated (1 nM Human Osteoblastoma Bone Cell Line UNI-ZAP ™ E2) fraction I HOS cell line XR H0292 Human OB HOS treated (10 nM Human Osteoblastoma Bone Cell Line UNI-ZAP ™ E2) fraction I HOS cell line XR H0293 WI 38 cells UNI-ZAP ™ XR H0294 Amniotic Cells - TNF induced Amniotic Cells - TNF Placenta Cell Line UNI-ZAP ™ induced XR H0295 Amniotic Cells - Primary Culture Amniotic Cells - Primary Placenta Cell Line UNI-ZAP ™ Culture XR H0298 HCBB''s differential CAMA1Ee Cell Line Breast Cell Line UNI-ZAP ™ consolidation XR H0299 HCBA''s differential CAMA1Ee Cell Line Breast Cell Line UNI-ZAP ™ consolidation XR H0300 CD34 positive cells (Cord Blood) CD34 Positive Cells Cord Blood ZAP EXPRESS ™ H0305 CD34 positive cells (Cord Blood) CD34 Positive Cells Cord Blood ZAP EXPRESS ™ H0306 CD34 depleted Buffy Coat (Cord CD34 Depleted Buffy Coat Cord Blood ZAP Blood) (Cord Blood) EXPRESS ™ H0309 Human Chronic Synovitis Synovium, Chronic Synovium disease UNI-ZAP ™ Synovitis/Osteoarthritis XR H0310 human caudate nucleus Brain Brain UNI-ZAP ™ XR H0313 human pleural cancer pleural cancer disease pBLUESCRIPT ™ H0316 HUMAN STOMACH Human Stomach Stomach UNI-ZAP ™ XR H0318 HUMAN B CELL LYMPHOMA Human B Cell Lymphoma Lymph Node disease UNI-ZAP ™ XR H0320 Human frontal cortex Human Frontal Cortex Brain UNI-ZAP ™ XR H0321 HUMAN SCHWANOMA Schwanoma Nerve disease UNI-ZAP ™ XR H0327 human corpus colosum Human Corpus Callosum Brain UNI-ZAP ™ XR H0328 human ovarian cancer Ovarian Cancer Ovary disease UNI-ZAP ™ XR H0329 Dermatofibrosarcoma Dermatofibrosarcoma Skin disease UNI-ZAP ™ Protuberance Protuberans XR H0330 HCBB''s Subtractive (-mito CAMA1Ee Cell Line Breast Cell Line UNI-ZAP ™ genes) XR H0331 Hepatocellular Tumor Hepatocellular Tumor Liver disease LAMBDA ZAP ™ II H0333 Hemangiopericytoma Hemangiopericytoma Blood vessel disease LAMBDA ZAP ™ II H0334 Kidney cancer Kidney Cancer Kidney disease UNI-ZAP ™ XR H0339 Duodenum Duodenum UNI-ZAP ™ XR H0340 Corpus Callosum Corpus Collosum-93052 UNI-ZAP ™ XR H0341 Bone Marrow Cell Line (RS4; 11) Bone Marrow Cell Line Bone Cell Line UNI-ZAP ™ RS4; 11 Marrow XR H0342 Lingual Gyrus Lingual Gyrus Brain UNI-ZAP ™ XR H0343 stomach cancer (human) Stomach Cancer - 5383A disease UNI-ZAP ™ (human) XR H0344 Adipose tissue (human) Adipose - 6825A (human) UNI-ZAP ™ XR H0345 SKIN Skin - 4000868H Skin UNI-ZAP ™ XR H0346 Brain-medulloblastoma Brain (Medulloblastoma)- Brain disease UNI-ZAP ™ 9405C006R XR H0349 human adult liver cDNA library Human Adult Liver Liver pCMVSport 1 H0350 Human Fetal Liver, mixed 10 & Human Fetal Liver, mixed Liver UNI-ZAP ™ 14 week 10&14 Week XR H0351 Glioblastoma Glioblastoma Brain disease UNI-ZAP ™ XR H0352 wilm''s tumor Wilm''s Tumor disease UNI-ZAP ™ XR H0354 Human Leukocytes Human Leukocytes Blood Cell Line pCMVSport 1 H0355 Human Liver Human Liver, normal Adult pCMVSport 1 H0356 Human Kidney Human Kidney Kidney pCMVSport 1 H0357 H. Normalized Fetal Liver, II Human Fetal Liver Liver UNI-ZAP ™ XR H0359 KMH2 cell line KMH2 ZAP EXPRESS ™ H0360 Hemangiopericytoma Hemangiopericytoma disease H0361 Human rejected kidney Human Rejected Kidney disease pBLUESCRIPT ™ H0362 HeLa cell line HELA CELL LINE pSport1 H0363 Human Brain Medulla, subtracted Human Brain Medulla pBLUESCRIPT ™ H0364 Human Osteoclastoma, excised Human Osteoclastoma disease pBLUESCRIPT ™ H0365 Osteoclastoma-normalized B Human Osteoclastoma disease UNI-ZAP ™ XR H0366 L428 cell line L428 ZAP EXPRESS ™ H0369 H. Atrophic Endometrium Atrophic Endometrium and UNI-ZAP ™ myometrium XR H0370 H. Lymph node breast Cancer Lymph node with Met. disease UNI-ZAP ™ Breast Cancer XR H0371 Eosinophils-Hypereosinophilia Eosinophils- disease UNI-ZAP ™ patient Hypereosinophilia patient XR H0372 Human Testes Human Testes Testis pCMVSport 1 H0373 Human Heart Human Adult Heart Heart pCMVSport 1 H0374 Human Brain Human Brain pCMVSport 1 H0375 Human Lung Human Lung pCMVSport 1 H0376 Human Spleen Human Adult Spleen Spleen pCMVSport 1 H0379 Human Tongue, frac 1 Human Tongue pSport1 H0380 Human Tongue, frac 2 Human Tongue pSport1 H0381 Bone Cancer Bone Cancer disease UNI-ZAP ™ XR H0383 Human Prostate BPH, re-excision Human Prostate BPH UNI-ZAP ™ XR H0384 Brain, Kozak Human Brain pCMVSport 1 H0385 H. Leukocytes, Kozak Human Leukocytes Blood Cell Line pCMVSport 1 H0386 Leukocyte and Lung; 4 screens Human Leukocytes Blood Cell Line pCMVSport 1 H0388 Human Rejected Kidney, 704 re- Human Rejected Kidney disease pBLUESCRIPT ™ excision H0389 H. Brain, X-Chromosome Human Brain pCMVSport 1 hybridization H0390 Human Amygdala Depression, re- Human Amygdala disease pBLUESCRIPT ™ excision Depression H0391 H. Meniingima, M6 Human Meningima brain pSport1 H0392 H. Meningima, M1 Human Meningima brain pSport1 H0393 Fetal Liver, subtraction II Human Fetal Liver Liver pBLUESCRIPT ™ H0394 A-14 cell line Redd-Sternberg cell ZAP EXPRESS ™ H0395 A1-CELL LINE Redd-Sternberg cell ZAP EXPRESS ™ H0396 L1 Cell line Redd-Sternberg cell ZAP EXPRESS ™ H0398 Human Newborn Bladder Human Newborn Bladder pBLUESCRIPT ™ H0399 Human Kidney Cortex, re-rescue Human Kidney Cortex LAMBDA ZAP ™ II H0400 Human Striatum Depression, re- Human Brain, Striatum Brain LAMBDA rescue Depression ZAP ™ II H0401 Human Pituitary, subtracted V Human Pituitary pBLUESCRIPT ™ H0402 CD34 depleted Buffy Coat (Cord CD34 Depleted Buffy Coat Cord Blood ZAP Blood), re-excision (Cord Blood) EXPRESS ™ H0403 H. Umbilical Vein Endothelial HUVE Cells Umbilical Cell Line UNI-ZAP ™ Cells, IL4 induced vein XR H0404 H. Umbilical Vein endothelial HUVE Cells Umbilical Cell Line UNI-ZAP ™ cells, uninduced vein XR H0405 Human Pituitary, subtracted VI Human Pituitary pBLUESCRIPT ™ H0406 H Amygdala Depression, Human Amygdala UNI-ZAP ™ subtracted Depression XR H0408 Human kidney Cortex, subtracted Human Kidney Cortex pBLUESCRIPT ™ H0409 H. Striatum Depression, Human Brain, Striatum Brain pBLUESCRIPT ™ subtracted Depression H0410 H. Male bladder, adult H Male Bladder, Adult Bladder pSport1 H0411 H Female Bladder, Adult Human Female Adult Bladder pSport1 Bladder H0412 Human umbilical vein endothelial HUVE Cells Umbilical Cell Line pSport1 cells, IL-4 induced vein H0413 Human Umbilical Vein HUVE Cells Umbilical Cell Line pSport1 Endothelial Cells, uninduced vein H0414 Ovarian Tumor I, OV5232 Ovarian Tumor, OV5232 Ovary disease pSport1 H0415 H. Ovarian Tumor, II, OV5232 Ovarian Tumor, OV5232 Ovary disease pCMVSport 2.0 H0416 Human Neutrophils, Activated, Human Neutrophil - Blood Cell Line pBLUESCRIPT ™ re-excision Activated H0417 Human Pituitary, subtracted VIII Human Pituitary pBLUESCRIPT ™ H0418 Human Pituitary, subtracted VII Human Pituitary pBLUESCRIPT ™ H0419 Bone Cancer, re-excision Bone Cancer UNI-ZAP ™ XR H0421 Human Bone Marrow, re-excision Bone Marrow pBLUESCRIPT ™ H0422 T-Cell PHA 16 hrs T-Cells Blood Cell Line pSport1 H0423 T-Cell PHA 24 hrs T-Cells Blood Cell Line pSport1 H0424 Human Pituitary, subt IX Human Pituitary pBLUESCRIPT ™ H0427 Human Adipose Human Adipose, left pSport1 hiplipoma H0428 Human Ovary Human Ovary Tumor Ovary pSport1 H0429 K562 + PMA (36 hrs), re-excision K562 Cell line cell line Cell Line ZAP EXPRESS ™ H0431 H. Kidney Medulla, re-excision Kidney medulla Kidney pBLUESCRIPT ™ H0432 H. Kidney Pyramid Kidney pyramids Kidney pBLUESCRIPT ™ H0433 Human Umbilical Vein HUVE Cells Umbilical Cell Line pBLUESCRIPT ™ Endothelial cells, frac B, re- vein excision H0434 Human Brain, striatum, re- Human Brain, Striatum pBLUESCRIPT ™ excision H0435 Ovarian Tumor 10-3-95 Ovarian Tumor, OV350721 Ovary pCMVSport 2.0 H0436 Resting T-Cell Library, II T-Cells Blood Cell Line pSport1 H0437 H Umbilical Vein Endothelial HUVE Cells Umbilical Cell Line LAMBDA Cells, frac A, re-excision vein ZAP ™ II H0438 H. Whole Brain #2, re-excision Human Whole Brain #2 ZAP EXPRESS ™ H0439 Human Eosinophils Eosinophils pBLUESCRIPT ™ H0440 FGF enriched mixed library Mixed libraries pCMVSport 1 H0441 H. Kidney Cortex, subtracted Kidney cortex Kidney pBLUESCRIPT ™ H0442 H. Striatum Depression, subt II Human Brain, Striatum Brain pBLUESCRIPT ™ Depression H0443 H. Adipose, subtracted Human Adipose, left pSport1 hiplipoma H0444 Spleen metastic melanoma Spleen, Metastic malignant Spleen disease pSport1 melanoma H0445 Spleen, Chronic lymphocytic Human Spleen, CLL Spleen disease pSport1 leukemia H0447 Salivary gland, re-excision Human Salivary Gland Salivary UNI-ZAP ™ gland XR H0448 Salivary gland, subtracted Human Salivary Gland Salivary LAMBDA gland ZAP ™ II H0449 CD34+ cell, I CD34 positive cells pSport1 H0450 CD34+cells, II CD34 positive cells pCMVSport 2.0 H0453 H. Kidney Pyramid, subtracted Kidney pyramids Kidney pBLUESCRIPT ™ H0455 H. Striatum Depression, subt Human Brain, Striatum Brain pBLUESCRIPT ™ Depression H0456 H Kidney Cortex, subtracted III Human Kidney Cortex pBLUESCRIPT ™ H0457 Human Eosinophils Human Eosinophils pSport1 H0458 CD34+ cell, I, frac II CD34 positive cells pSport1 H0459 CD34+cells, II, FRACTION 2 CD34 positive cells pCMVSport 2.0 H0461 H. Kidney Medulla, subtracted Kidney medulla Kidney pBLUESCRIPT ™ H0462 H. Amygdala Depression, Brain pBLUESCRIPT ™ subtracted H0477 Human Tonsil, Lib 3 Human Tonsil Tonsil pSport1 H0478 Salivary Gland, Lib 2 Human Salivary Gland Salivary pSport1 gland H0479 Salivary Gland, Lib 3 Human Salivary Gland Salivary pSport1 gland H0480 L8 cell line L8 cell line ZAP EXPRESS ™ H0483 Breast Cancer cell line, MDA 36 Breast Cancer Cell line, pSport1 MDA 36 H0484 Breast Cancer Cell line, Breast Cancer Cell line, pSport1 angiogenic Angiogenic, 36T3 H0485 Hodgkin''s Lymphoma I Hodgkin''s Lymphoma I disease pCMVSport 2.0 H0486 Hodgkin''s Lymphoma II Hodgkin''s Lymphoma II disease pCMVSport 2.0 H0487 Human Tonsils, lib I Human Tonsils pCMVSport 2.0 H0488 Human Tonsils, Lib 2 Human Tonsils pCMVSport 2.0 H0489 Crohn''s Disease Ileum Intestine disease pSport1 H0490 Hl-60, untreated, subtracted Human HL-60 Cells, Blood Cell Line UNI-ZAP ™ unstimulated XR H0491 HL-60, PMA 4 H, subtracted HL-60 Cells, PMA Blood Cell Line UNI-ZAP ™ stimulated 4 H XR H0492 HL-60, RA 4 h, Subtracted HL-60 Cells, RA stimulated Blood Cell Line UNI-ZAP ™ for 4 H XR H0493 HL-60, PMA 1 d, subtracted HL-60 Cells, PMA Blood Cell Line UNI-ZAP ™ stimulated for 1 day XR H0494 Keratinocyte Keratinocyte pCMVSport 2.0 H0497 HEL cell line HEL cell line HEL 92.1.7 pSport1 H0505 Human Astrocyte Human Astrocyte pSport1 H0506 Ulcerative Colitis Colon Colon pSport1 H0509 Liver, Hepatoma Human Liver, Hepatoma, Liver disease pCMVSport 3.0 patient 8 H0510 Human Liver, normal Human Liver, normal, Liver pCMVSport 3.0 Patient # 8 H0512 Keratinocyte, lib 3 Keratinocyte pCMVSport 2.0 H0517 Nasal polyps Nasal polyps pCMVSport 2.0 H0518 pBMC stimulated w/ poly I/C pBMC stimulated with poly pCMVSport 3.0 I/C H0519 NTERA2, control NTERA2, Teratocarcinoma pCMVSport 3.0 cell line H0520 NTERA2 + retinoic acid, 14 days NTERA2, Teratocarcinoma pSport1 cell line H0521 Primary Dendritic Cells, lib 1 Primary Dendritic cells pCMVSport 3.0 H0522 Primary Dendritic cells, frac 2 Primary Dendritic cells pCMVSport 3.0 H0523 Primary Dendritic Primary Dendritic cells pSport1 cells, CapFinder2, frac 1 H0524 Primary Dendritic Cells, Primary Dendritic cells pSport1 CapFinder, frac 2 H0525 PCR, pBMC I/C treated pBMC stimulated with poly pCR II I/C [Invitrogen] H0528 Poly[I]/Poly[C] Normal Lung Poly[I]/Poly[C] Normal pCMVSport 3.0 Fibroblasts Lung Fibroblasts H0529 Myoloid Progenitor Cell Line TF-1 Cell Line; Myoloid pCMVSport 3.0 progenitor cell line H0530 Human Dermal Endothelial Human Dermal Endothelial pSport1 Cells, untreated Cells; untreated H0533 Human Stromal endometrial Human Stromal pSport1 fibroblasts, treated w/ estradiol endometrial fibroblasts, treated wit H0535 Human ovary tumor cell Ovarian Tumor, OV350721 Ovary disease pSport1 OV350721 H0537 H. Primary Dendritic Cells, lib 3 Primary Dendritic cells pCMVSport 2.0 H0538 Merkel Cells Merkel cells Lymph node pSport1 H0539 Pancreas Islet Cell Tumor Pancreas Islet Cell Tumour Pancreas disease pSport1 H0540 Skin, burned Skin, leg burned Skin pSport1 H0542 T Cell helper I Helper T cell pCMVSport 3.0 H0543 T cell helper II Helper T cell pCMVSport 3.0 H0544 Human endometrial stromal cells Human endometrial stromal pCMVSport 3.0 cells H0545 Human endometrial stromal cells- Human endometrial stromal pCMVSport 3.0 treated with progesterone cells-treated with proge H0546 Human endometrial stromal cells- Human endometrial stromal pCMVSport 3.0 treated with estradiol cells-treated with estra H0547 NTERA2 teratocarcinoma cell NTERA2, Teratocarcinoma pSport1 line + retinoic acid (14 days) cell line H0548 Human Skin Fibroblasts, normal Human Skin Fibroblasts pBLUESCRIPT ™ H0549 H. Epididiymus, caput & corpus Human Epididiymus, caput UNI-ZAP ™ and corpus XR H0550 H. Epididiymus, cauda Human Epididiymus, cauda UNI-ZAP ™ XR H0551 Human Thymus Stromal Cells Human Thymus Stromal pCMVSport 3.0 Cells H0552 Signal trap, Femur Bone Femur Bone marrow, Other Marrow, pooled pooled from 8 male/female H0553 Human Placenta Human Placenta pCMVSport 3.0 H0555 Rejected Kidney, lib 4 Human Rejected Kidney Kidney disease pCMVSport 3.0 H0556 Activated T- T-Cells Blood Cell Line UNI-ZAP ™ cell(12 h)/Thiouridine-re-excision XR H0559 HL-60, PMA 4 H, re-excision HL-60 Cells, PMA Blood Cell Line UNI-ZAP ™ stimulated 4 H XR H0560 KMH2 KMH2 pCMVSport 3.0 H0561 L428 L428 pCMVSport 3.0 H0562 Human Fetal Brain, normalized Human Fetal Brain pCMVSport 2.0 c5-11-26 H0563 Human Fetal Brain, normalized Human Fetal Brain pCMVSport 2.0 50021F H0564 Human Fetal Brain, normalized Human Fetal Brain pCMVSport 2.0 C5001F H0565 Human Fetal Brain, normalized Human Fetal Brain pCMVSport 2.0 100024F H0566 Human Fetal Brain, normalized Human Fetal Brain pCMVSport 2.0 c50F H0567 Human Fetal Brain, normalized Human Fetal Brain pCMVSport 2.0 A5002F H0569 Human Fetal Brain, normalized Human Fetal Brain pCMVSport 2.0 CO H0570 Human Fetal Brain, normalized Human Fetal Brain pCMVSport 2.0 C500H H0571 Human Fetal Brain, normalized Human Fetal Brain pCMVSport 2.0 C500HE H0572 Human Fetal Brain, normalized Human Fetal Brain pCMVSport 2.0 AC5002 H0574 Hepatocellular Tumor; re- Hepatocellular Tumor Liver disease LAMBDA excision ZAP ™ II H0575 Human Adult Pulmonary; re- Human Adult Pulmonary Lung UNI-ZAP ™ excision XR H0576 Resting T-Cell; re-excision T-Cells Blood Cell Line LAMBDA ZAP ™ II H0578 Human Fetal Thymus Fetal Thymus Thymus pSport1 H0579 Pericardium Pericardium Heart pSport1 H0580 Dendritic cells, pooled Pooled dendritic cells pCMVSport 3.0 H0581 Human Bone Marrow, treated Human Bone Marrow Bone pCMVSport 3.0 Marrow H0583 B Cell lymphoma B Cell Lymphoma B Cell disease pCMVSport 3.0 H0584 Activated T-cells, 24 hrs, re- Activated T-Cells Blood Cell Line UNI-ZAP ™ excision XR H0585 Activated T-Cells, 12 hrs, re- Activated T-Cells Blood Cell Line UNI-ZAP ™ excision XR H0586 Healing groin wound, 6.5 hours healing groin wound, 6.5 groin disease pCMVSport 3.0 post incision hours post incision-2/ H0587 Healing groin wound; 7.5 hours Groin-Feb. 19, 1997 groin disease pCMVSport 3.0 post incision H0589 CD34 positive cells (cord CD34 Positive Cells Cord Blood ZAP blood), re-ex EXPRESS ™ H0590 Human adult small intestine, re- Human Adult Small Small Int. UNI-ZAP ™ excision Intestine XR H0591 Human T-cell lymphoma; re- T-Cell Lymphoma T-Cell disease UNI-ZAP ™ excision XR H0592 Healing groin wound - zero hr HGS wound healing disease pCMVSport 3.0 post-incision (control) project; abdomen H0593 Olfactory epithelium; nasalcavity Olfactory epithelium from pCMVSport 3.0 roof of left nasal cacit H0594 Human Lung Cancer; re-excision Human Lung Cancer Lung disease LAMBDA ZAP ™ II H0595 Stomach cancer (human); re- Stomach Cancer - 5383A disease UNI-ZAP ™ excision (human) XR H0596 Human Colon Cancer; re-excision Human Colon Cancer Colon LAMBDA ZAP ™ II H0597 Human Colon; re-excision Human Colon LAMBDA ZAP ™ II H0598 Human Stomach; re-excision Human Stomach Stomach UNI-ZAP ™ XR H0599 Human Adult Heart; re-excision Human Adult Heart Heart UNI-ZAP ™ XR H0600 Healing Abdomen wound; Abdomen disease pCMVSport 3.0 70&90 min post incision H0601 Healing Abdomen Wound; 15 Abdomen disease pCMVSport 3.0 days post incision H0602 Healing Abdomen Wound; 21&29 Abdomen disease pCMVSport 3.0 days post incision H0604 Human Pituitary, re-excision Human Pituitary pBLUESCRIPT ™ H0606 Human Primary Breast Cancer; re- Human Primary Breast Breast disease UNI-ZAP ™ excision Cancer XR H0607 H. Leukocytes, normalized cot H. Leukocytes pCMVSport 1 50A3 H0608 H. Leukocytes, control H. Leukocytes pCMVSport 1 H0609 H. Leukocytes, normalized cot H. Leukocytes pCMVSport 1 >500A H0610 H. Leukocytes, normalized cot H. Leukocytes pCMVSport 1 5A H0611 H. Leukocytes, normalized cot H. Leukocytes pCMVSport 1 500 B H0612 H. Leukocytes, normalized cot 50 B H. Leukocytes pCMVSport 1 H0613 H. Leukocytes, normalized cot 5B H. Leukocytes pCMVSport 1 H0614 H. Leukocytes, normalized cot H. Leukocytes pCMVSport 1 500 A H0615 Human Ovarian Cancer Ovarian Cancer Ovary disease UNI-ZAP ™ Reexcision XR H0616 Human Testes, Reexcision Human Testes Testis UNI-ZAP ™ XR H0617 Human Primary Breast Cancer Human Primary Breast Breast disease UNI-ZAP ™ Reexcision Cancer XR H0618 Human Adult Testes, Large Human Adult Testis Testis UNI-ZAP ™ Inserts, Reexcision XR H0619 Fetal Heart Human Fetal Heart Heart UNI-ZAP ™ XR H0620 Human Fetal Kidney; Reexcision Human Fetal Kidney Kidney UNI-ZAP ™ XR H0622 Human Pancreas Tumor; Human Pancreas Tumor Pancreas disease UNI-ZAP ™ Reexcision XR H0623 Human Umbilical Vein; Human Umbilical Vein Umbilical UNI-ZAP ™ Reexcision Endothelial Cells vein XR H0624 12 Week Early Stage Human II; Twelve Week Old Early Embryo UNI-ZAP ™ Reexcision Stage Human XR H0625 Ku 812F Basophils Line Ku 812F Basophils pSport1 H0626 Saos2 Cells; Untreated Saos2 Cell Line; Untreated pSport1 H0627 Saos2 Cells; Vitamin D3 Treated Saos2 Cell Line; Vitamin pSport1 D3 Treated H0628 Human Pre-Differentiated Human Pre-Differentiated UNI-ZAP ™ Adipocytes Adipocytes XR H0629 Human Leukocyte, control #2 Human Normalized pCMVSport 1 leukocyte H0630 Human Leukocytes, normalized Human Normalized pCMVSport 1 control #4 leukocyte H0631 Saos2, Dexamethosome Treated Saos2 Cell Line; pSport1 Dexamethosome Treated H0632 Hepatocellular Tumor; re-excision Hepatocellular Tumor Liver LAMBDA ZAP ™ II H0633 Lung Carcinoma A549 TNFalpha TNFalpha activated A549-- disease pSport1 activated Lung Carcinoma H0634 Human Testes Tumor, re-excision Human Testes Tumor Testis disease UNI-ZAP ™ XR H0635 Human Activated T-Cells, re- Activated T-Cells Blood Cell Line UNI-ZAP ™ excision XR H0637 Dendritic Cells From CD34 Cells Dentritic cells from CD34 pSport1 cells H0638 CD40 activated monocyte CD40 activated monocyte pSport1 dendridic cells dendridic cells H0639 Ficolled Human Stromal Cells, Ficolled Human Stromal Other 5Fu treated Cells, 5Fu treated H0640 FICOLL ™ ed Human Stromal FICOLL ™ ed Human Other Cells, Untreated Stromal Cells, Untreated H0641 LPS activated derived dendritic LPS activated monocyte pSport1 cells derived dendritic cells H0642 Hep G2 Cells, lambda library Hep G2 Cells Other H0643 Hep G2 Cells, PCR library Hep G2 Cells Other H0644 Human Placenta (re-excision) Human Placenta Placenta UNI-ZAP ™ XR H0645 Fetal Heart, re-excision Human Fetal Heart Heart UNI-ZAP ™ XR H0646 Lung, Cancer (4005313 A3): Metastatic squamous cell pSport1 Invasive Poorly Differentiated lung carcinoma, poorly di Lung Adenocarcinoma, H0647 Lung, Cancer (4005163 B7): Invasive poorly disease pSport1 Invasive, Poorly Diff. differentiated lung Adenocarcinoma, Metastatic adenocarcinoma H0648 Ovary, Cancer: (4004562 B6) Papillary Cstic neoplasm of disease pSport1 Papillary Serous Cystic low malignant potentia Neoplasm, Low Malignant Pot H0649 Lung, Normal: (4005313 B1) Normal Lung pSport1 H0650 B-Cells B-Cells pCMVSport 3.0 H0651 Ovary, Normal: (9805C040R) Normal Ovary pSport1 H0652 Lung, Normal: (4005313 B1) Normal Lung pSport1 H0653 Stromal Cells Stromal Cells pSport1 H0654 Lung, Cancer: (4005313 A3) Metastatic Squamous cell Other Invasive Poorly-differentiated lung Carcinoma poorly dif Metastatic lung adenoc H0656 B-cells (unstimulated) B-cells (unstimulated) pSport1 H0657 B-cells (stimulated) B-cells (stimulated) pSport1 H0658 Ovary, Cancer (9809C332): 9809C332-Poorly Ovary & disease pSport1 Poorly differentiated differentiate Fallopian adenocarcinoma Tubes H0659 Ovary, Cancer (15395A1F): Grade II Papillary Ovary disease pSport1 Grade II Papillary Carcinoma Carcinoma, Ovary H0660 Ovary, Cancer: (15799A1F) Poorly differentiated disease pSport1 Poorly differentiated carcinoma carcinoma, ovary H0661 Breast, Cancer: (4004943 A5) Breast cancer disease pSport1 H0662 Breast, Normal: (4005522B2) Normal Breast - Breast pSport1 #4005522(B2) H0663 Breast, Cancer: (4005522 A2) Breast Cancer - Breast disease pSport1 #4005522(A2) H0664 Breast, Cancer: (9806C012R) Breast Cancer Breast disease pSport1 H0665 Stromal cells 3.88 Stromal cells 3.88 pSport1 H0666 Ovary, Cancer: (4004332 A2) Ovarian Cancer, Sample disease pSport1 #4004332A2 H0667 Stromal cells(HBM3.18) Stromal cell(HBM 3.18) pSport1 H0668 stromal cell clone 2.5 stromal cell clone 2.5 pSport1 H0669 Breast, Cancer: (4005385 A2) Breast Cancer (4005385A2) Breast pSport1 H0670 Ovary, Cancer(4004650 A3): Ovarian Cancer - pSport1 Well-Differentiated 4004650A3 Micropapillary Serous Carcinoma H0671 Breast, Cancer: (9802C02OE) Breast Cancer -Sample # pSport1 9802C02OE H0672 Ovary, Cancer: (4004576 A8) Ovarian Ovary pSport1 Cancer(4004576A8) H0673 Human Prostate Cancer, Stage Human Prostate Cancer, Prostate UNI-ZAP ™ B2; re-excision stage B2 XR H0674 Human Prostate Cancer, Stage C; Human Prostate Cancer, Prostate UNI-ZAP ™ re-excission stage C XR H0675 Colon, Cancer: (9808C064R) Colon Cancer 9808C064R pCMVSport 3.0 H0676 Colon, Cancer: (9808C064R)- Colon Cancer 9808C064R pCMVSport 3.0 total RNA H0677 TNFR degenerate oligo B-Cells pCR II [Invitrogen] H0678 screened clones from placental Placenta Placenta Other library H0679 screened clones from Tonsil Human Tonsils Other library H0682 Serous Papillary Adenocarcinoma serous papillary pCMVSport 3.0 adenocarcinoma (9606G304SPA3B) H0683 Ovarian Serous Papillary Serous papillary pCMVSport 3.0 Adenocarcinoma adenocarcinoma, stage 3C (9804G01 H0684 Serous Papillary Adenocarcinoma Ovarian Cancer-9810G606 Ovaries pCMVSport 3.0 H0685 Adenocarcinoma of Ovary, Adenocarcinoma of Ovary, pCMVSport 3.0 Human Cell Line, # OVCAR-3 Human Cell Line, # OVCAR- H0686 Adenocarcinoma of Ovary, Adenocarcinoma of Ovary, pCMVSport 3.0 Human Cell Line Human Cell Line, # SW- 626 H0687 Human normal Human normal Ovary pCMVSport 3.0 ovary(#9610G215) ovary(#9610G215) H0688 Human Ovarian Human Ovarian pCMVSport 3.0 Cancer(#9807G017) cancer(#9807G017), mRNA from Maura Ru H0689 Ovarian Cancer Ovarian Cancer, pCMVSport 3.0 #9806G019 H0690 Ovarian Cancer, # 9702G001 Ovarian Cancer, pCMVSport 3.0 #9702G001 H0691 Normal Ovary, #9710G208 normal ovary, #9710G208 pCMVSport 3.0 H0692 BLyS Receptor from Expression B Cell Lymphoma B Cell pCMVSport 3.0 Cloning H0693 Normal Prostate #ODQ3958EN Normal Prostate Tissue # pCMVSport 3.0 ODQ3958EN H0694 Prostate gland adenocarcinoma Prostate gland, prostate pCMVSport 3.0 adenocarcinoma, mod/diff, gland gleason H0695 mononucleocytes from patient mononucleocytes from pCMVSport 3.0 patient at Shady Grove Hospit L0002 Atrium cDNA library Human heart L0004 CLONTECH ™ HL 1065a L0005 CLONTECH ™ human aorta polyA+ mRNA (#6572) L0009 EST from 8p21.3-p22 L0012 HDMEC cDNA library L0015 Human L0017 Human (J. Swensen) L0018 Human (M. Lovett) L0021 Human adult (K. Okubo) L0022 Human adult lung 3″ directed MboI cDNA L0023 human adult testis L0024 Human brain ARSanders L0025 Human brain striatum L0032 Human chromosome 12p cDNAs L0033 Human chromosome 13q14 cDNA L0034 Human chromosome 14 L0038 Human chromosome 6 L0040 Human colon mucosa L0041 Human epidermal keratinocyte L0045 Human keratinocyte differential display (B. Lin) L0051 Human mRNA (Tripodis and Ragoussis) L0052 Human normalized K562-cDNA L0053 Human pancreatic tumor L0055 Human promyelocyte L0060 Human thymus NSTH II L0065 Liver HepG2 cell line. L0070 Selected chromosome 21 cDNA library L0096 Subtracted human retina L0097 Subtracted human retinal pigment epithelium (RPE) L0103 DKFZphamy1 amygdala L0105 Human aorta polyA+ (TFujiwara) aorta L0109 Human brain cDNA brain L0114 Human fetal brain (R. L. Margolis) brain L0117 Human fetal brain cDNA brain (T. M. Gress) L0118 Human fetal brain S. Meier-Ewert brain L0121 Stratagene catalog #936206 brain L0126 Human fibroblast cDNA fibroblast L0130 Human hippocampus, Stratagene hippocampus catalog #936205 L0136 Human neuroepithelium neuroepithelium (N. Jiang) L0138 Human normal gingiva normal gingiva L0140 Human pancreatic cancer pancreatic cancer (CWallrapp) L0141 Human pancreatic islet cell pancreatic islet L0142 Human placenta cDNA placenta (TFujiwara) L0143 Human placenta polyA+ placenta (TFujiwara) L0145 Human retina (D. Swanson) retina L0146 Human fovea cDNA retinal fovea L0149 DKFZphsnu1 subthalamic nucleus L0151 Human testis (C. De Smet) testis L0157 Human fetal brain (TFujiwara) brain L0158 Human fetal brain QBoqin brain L0162 Human brain frontal cortex frontal cortex brain L0163 Human heart cDNA heart (YNakamura) L0171 Human lung adenocarcinoma lung adenocarcinoma A549 A549 L0175 Human retina cell line ARPE-19 retina ARPE-19 L0177 Human newborn melanocytes Clonetics (T. Vogt) Corp. (San Diego, CA) strain #68 and 2486 L0182 Human HeLa (Y. Wang) HeLa L0183 Human HeLa cells (M. Lovett) HeLa L0185 Human immortalized fibroblasts HS74 and its (H. L. Ozer) SV40- transformed sublines L0186 Human salivary gland cell line salivary gland HSG HSG L0187 Human fibrosarcoma cell line fibrosarcoma HT1080 HT1080 L0194 Human pancreatic cancer cell line pancreatic cancer Patu 8988t Patu 8988t L0295 Human liver EST (Y. L. Yu) liver L0307 Human C3-A11N C3-A11N; clonally related variant of OCI LY8-C3P L0309 Human E8CASS breast adenocarcinoma E8CASS; variant of MCF7 L0351 Infant brain, Bento Soares BA, M13- derived L0352 Normalized infant brain, Bento BA, M13- Soares derived L0353 21q Placenta, F. Tassone and K. pBLUESCRIPT ™ Gardiner L0354 JG, Human foetal Kidney tissue pBLUESCRIPT ™ L0355 P, Human foetal Brain Whole pBLUESCRIPT ™ tissue L0356 S, Human foetal Adrenals tissue pBLUESCRIPT ™ L0357 V, Human Placenta tissue Bluescript KS II+ L0361 STRATAGENE ™ ovary ovary pBLUESCRIPT ™ (#937217) SK L0362 STRATAGENE ™ ovarian cancer pBLUESCRIPT ™ (#937219) SK− L0363 NCI_CGAP_GC2 germ cell tumor pBLUESCRIPT ™ SK− L0364 NCI_CGAP_GC5 germ cell tumor pBLUESCRIPT ™ SK− L0365 NCI_CGAP_Phe1 pheochromocytoma pBLUESCRIPT ™ SK− L0366 STRATAGENE ™ schizo brain schizophrenic brain S-11 pBLUESCRIPT ™ S11 frontal lobe SK− L0367 NCI_CGAP_Sch1 Schwannoma tumor pBLUESCRIPT ™ SK− L0368 NCI_CGAP_SS1 synovial sarcoma pBLUESCRIPT ™ SK− L0369 NCI_CGAP_AA1 adrenal adenoma adrenal gland pBLUESCRIPT ™ SK− L0370 Johnston frontal cortex pooled frontal lobe brain pBLUESCRIPT ™ SK− L0371 NCI_CGAP_Br3 breast tumor breast pBLUESCRIPT ™ SK− L0372 NCI_CGAP_Co12 colon tumor colon pBLUESCRIPT ™ SK− L0373 NCI_CGAP_Co11 tumor colon pBLUESCRIPT ™ SK− L0374 NCI_CGAP_Co2 tumor colon pBLUESCRIPT ™ SK− L0375 NCI_CGAP_Kid6 kidney tumor kidney pBLUESCRIPT ™ SK− L0376 NCI_CGAP_Lar1 larynx larynx pBLUESCRIPT ™ SK− L0377 NCI_CGAP_HN2 squamous cell carcinoma larynx pBLUESCRIPT ™ from vocal cord SK− L0378 NCI_CGAP_Lu1 lung tumor lung pBLUESCRIPT ™ SK− L0379 NCI_CGAP_Lym3 lymphoma lymph node pBLUESCRIPT ™ SK− L0380 NCI_CGAP_HN1 squamous cell carcinoma lymph node pBLUESCRIPT ™ SK− L0381 NCI_CGAP_HN4 squamous cell carcinoma pharynx pBLUESCRIPT ™ SK− L0382 NCI_CGAP_Pr25 epithelium (cell line) prostate pBLUESCRIPT ™ SK− L0383 NCI_CGAP_Pr24 invasive tumor (cell line) prostate pBLUESCRIPT ™ SK− L0384 NCI_CGAP_Pr23 prostate tumor prostate pBLUESCRIPT ™ SK− L0385 NCI_CGAP_Gas1 gastric tumor stomach pBLUESCRIPT ™ SK− L0386 NCI_CGAP_HN3 squamous cell carcinoma tongue pBLUESCRIPT ™ from base of tongue SK− L0387 NCI_CGAP_GCB0 germinal center B-cells tonsil pBLUESCRIPT ™ SK− L0388 NCI_CGAP_HN6 normal gingiva (cell line pBLUESCRIPT ™ from immortalized kerati SK− L0389 NCI_CGAP_HN5 normal gingiva (cell line pBLUESCRIPT ™ from primary keratinocyt SK− L0393 B, Human Liver tissue gt11 L0394 H, Human adult Brain Cortex gt11 tissue L0404 b4HB3MA Cot109 + 103 + 85-Bio Lafmid A L0405 b4HB3MA Cot109 + 103-Bio Lafmid A L0411 1-NIB Lafmid BA L0414 b4HB3MA Lafmid BA L0415 b4HB3MA Cot8-HAP-Ft Lafmid BA L0416 b4HB3MA-Cot0.38-HAP-B Lafmid BA L0417 b4HB3MA-Cot0.38-HAP-Ft-6 Lafmid BA L0418 b4HB3MA-Cot109 + 10-Bio Lafmid BA L0419 b4HB3MA-Cot109 + 103 + 85-Bio Lafmid BA L0422 b4HB3MA-Cot12-HAP-B Lafmid BA L0424 b4HB3MA-Cot14.5 Lafmid BA L0425 b4HB3MA-Cot18-Bio Lafmid BA L0426 b4HB3MA-Cot51.5-HAP-Ft Lafmid BA L0427 b4HB3MA-FT20%-Biotin Lafmid BA L0428 Cot1374Ft-4HB3MA Lafmid BA L0430 Cot250Ft-b4HB3MA Lafmid BA L0433 HWM42YA Lafmid BA L0434 Infant brain library of Dr. M. Soares Lafmid BA L0435 Infant brain, LLNL array of Dr. Lafmid BA M. Soares 1NIB L0437 N-b4HB3MA-Cot109 Lafmid BA L0438 normalized infant brain cDNA total brain brain Lafmid BA L0439 Soares infant brain 1NIB whole brain Lafmid BA L0441 2HB3MK Lafmid BK L0442 4HB3MK Lafmid BK L0443 b4HB3MK Lafmid BK L0446 N4HB3MK Lafmid BK L0447 NHB3MK Lafmid BK L0448 3HFLSK20 Lafmid K L0451 N3HFLSK20 Lafmid K L0453 BATM1 lambda gt10 L0454 CLONTECH ™ adult human fat lambda gt10 cell library HL1108A L0455 Human retina cDNA randomly retina eye lambda gt10 primed sublibrary L0456 Human retina cDNA Tsp509I- retina eye lambda gt10 cleaved sublibrary L0457 multi-tissue normalized short- multi-tissue pooled lambda gt10 fragment L0459 Adult heart, CLONTECH ™ Lambda gt11 L0460 Adult heart, Lambda gt11 Lambda gt11 L0462 WATM1 lambda gt11 L0463 fetal brain cDNA brain brain lambda gt11 L0465 TEST1, Human adult Testis lambda nm1149 tissue L0467 Fetal heart, Lambda ZAP Express Lambda ZAP L0468 HE6W lambda zap L0469 T, Human adult Lambda Zap Rhabdomyosarcoma cell-line L0470 BL29 Burkitt''s lymphoma, LAMBDA Pascalis Sideras ZAP ™ II L0471 Human fetal heart, LAMBDA LAMBDA ZAP ™ Express ZAP ™ Express (STRATAGENE ™) L0475 KG1-a Lambda Zap Express KG1-a Lambda Zap cDNA library Express (STRATAGENE ™) L0476 Fetal brain, STRATAGENE ™ LAMBDA ZAP ™ II L0477 HPLA CCLee placenta LAMBDA ZAP ™ II L0480 STRATAGENE ™ cat#937212 Lambda ZAP, (1992) pBLUESCRIPT ™ SK− L0481 CD34+DIRECTIONAL LAMBDA ZAP ™ II L0482 HT29M6 LAMBDA ZAP ™ II L0483 Human pancreatic islet LAMBDA ZAP ™ II L0485 STRATAGENE ™ Human skeletal muscle leg muscle LAMBDA skeletal muscle cDNA library, ZAP ™ II cat. #936215. L0486 Human promyelocytic HL60 cell promyelocytic LAMBDA line (S. Herblot) HL60 cell line ZAP ™ II L0487 Human peripheral blood (Steve whole peripheral blood Lambda-Yes Elledge) L0492 Human Genomic pAMP L0493 NCI_CGAP_Ov26 papillary serous carcinoma ovary pAMP1 L0497 NCI_CGAP_HSC4 CD34+, CD38− from bone marrow pAMP1 normal bone marrow donor L0498 NCI_CGAP_HSC3 CD34+, T negative, patient bone marrow pAMP1 with chronic myelogenou L0499 NCI_CGAP_HSC2 stem cell 34+/38+ bone marrow pAMP1 L0500 NCI_CGAP_Brn20 oligodendroglioma brain pAMP1 L0501 NCI_CGAP_Brn21 oligodendroglioma brain pAMP1 L0502 NCI_CGAP_Br15 adenocarcinoma breast pAMP1 L0503 NCI_CGAP_Br17 adenocarcinoma breast pAMP1 L0504 NCI_CGAP_Br13 breast carcinoma in situ breast pAMP1 L0505 NCI_CGAP_Br12 invasive carcinoma breast pAMP1 L0506 NCI_CGAP_Br16 lobullar carcinoma in situ breast pAMP1 L0507 NCI_CGAP_Br14 normal epithelium breast pAMP1 L0508 NCI_CGAP_Lu25 bronchioalveolar carcinoma lung pAMP1 L0509 NCI_CGAP_Lu26 invasive adenocarcinoma lung pAMP1 L0510 NCI_CGAP_Ov33 borderline ovarian ovary pAMP1 carcinoma L0511 NCI_CGAP_Ov34 borderline ovarian ovary pAMP1 carcinoma L0512 NCI_CGAP_Ov36 borderline ovarian ovary pAMP1 carcinoma L0513 NCI_CGAP_Ov37 early stage papillary serous ovary pAMP1 carcinoma L0514 NCI_CGAP_Ov31 papillary serous carcinoma ovary pAMP1 L0515 NCI_CGAP_Ov32 papillary serous carcinoma ovary pAMP1 L0516 Chromosome 19p12-p13.1 exon pAMP10 L0517 NCI_CGAP_Pr1 pAMP10 L0518 NCI_CGAP_Pr2 pAMP10 L0519 NCI_CGAP_Pr3 pAMP10 L0520 NCI_CGAP_Alv1 alveolar pAMP10 rhabdomyosarcoma L0521 NCI_CGAP_Ew1 Ewing''s sarcoma pAMP10 L0522 NCI_CGAP_Kid1 kidney pAMP10 L0523 NCI_CGAP_Lip2 liposarcoma pAMP10 L0524 NCI_CGAP_Li1 liver pAMP10 L0525 NCI_CGAP_Li2 liver pAMP10 L0526 NCI_CGAP_Pr12 metastatic prostate bone pAMP10 lesion L0527 NCI_CGAP_Ov2 ovary pAMP10 L0528 NCI_CGAP_Pr5 prostate pAMP10 L0529 NCI_CGAP_Pr6 prostate pAMP10 L0530 NCI_CGAP_Pr8 prostate pAMP10 L0531 NCI_CGAP_Pr20 prostate metastasis, liver pAMP10 L0532 NCI_CGAP_Thy1 thyroid pAMP10 L0533 NCI_CGAP_HSC1 stem cells bone marrow pAMP10 L0534 Chromosome 7 Fetal Brain cDNA brain brain pAMP10 Library L0535 NCI_CGAP_Br5 infiltrating ductal breast pAMP10 carcinoma L0536 NCI_CGAP_Br4 normal ductal tissue breast pAMP10 L0537 NCI_CGAP_Ov6 normal cortical stroma ovary pAMP10 L0538 NCI_CGAP_Ov5 normal surface epithelium ovary pAMP10 L0539 Chromosome 7 Placental cDNA placenta pAMP10 Library L0540 NCI_CGAP_Pr10 invasive prostate tumor prostate pAMP10 L0541 NCI_CGAP_Pr7 low-grade prostatic prostate pAMP10 neoplasia L0542 NCI_CGAP_Pr11 normal prostatic epithelial prostate pAMP10 cells L0543 NCI_CGAP_Pr9 normal prostatic epithelial prostate pAMP10 cells L0544 NCI_CGAP_Pr4 prostatic intraepithelial prostate pAMP10 neoplasia - high grade L0545 NCI_CGAP_Pr4.1 prostatic intraepithelial prostate pAMP10 neoplasia - high grade L0546 NCI_CGAP_Pr18 stroma prostate pAMP10 L0547 NCI_CGAP_Pr16 tumor prostate pAMP10 L0548 Chromosome 7 Thymus cDNA thymus thymus pAMP10 Library L0549 NCI_CGAP_HN10 carcinoma in situ from pAMP10 retromolar trigone L0550 NCI_CGAP_HN9 normal squamous pAMP10 epithelium from retromolar trigone L0551 NCI_CGAP_HN7 normal squamous pAMP10 epithelium, floor of mouth L0552 NCI_CGAP_HN8 well-differentiated invasive pAMP10 carcinoma, floor of m L0553 NCI_CGAP_Co22 colonic adenocarcinoma colon pAMP10 L0554 NCI_CGAP_Li8 liver pAMP10 L0555 NCI_CGAP_Lu34 large cell carcinoma lung pAMP10 L0556 NCI_CGAP_Lu34.1 large cell carcinoma lung pAMP10 L0557 NCI_CGAP_Lu21 small cell carcinoma lung pAMP10 L0558 NCI_CGAP_Ov40 endometrioid ovarian ovary pAMP10 metastasis L0559 NCI_CGAP_Ov39 papillary serous ovarian ovary pAMP10 metastasis L0560 NCI_CGAP_HN12 moderate to poorly tongue pAMP10 differentiated invasive carcino L0561 NCI_CGAP_HN11 normal squamous tongue pAMP10 epithelium L0562 Chromosome 7 HeLa cDNA HeLa cell line; pAMP10 Library ATCC ™ L0563 Human Bone Marrow Stromal bone marrow pBLUESCRIPT ™ Fibroblast L0564 Jia bone marrow stroma bone marrow stroma pBLUESCRIPT ™ L0565 Normal Human Trabecular Bone Bone Hip pBLUESCRIPT ™ Cells L0579 Human fetal brain QBoqin2 cerebrum and cerebellum pBLUESCRIPT ™ SK L0581 STRATAGENE ™ liver liver pBLUESCRIPT ™ (#937224) SK L0583 STRATAGENE ™ cDNA library pBLUESCRIPT ™ Human fibroblast, cat#937212 SK L0584 STRATAGENE ™ cDNA library pBLUESCRIPT ™ Human heart, cat#936208 SK L0586 HTCDL1 pBLUESCRIPT ™ SK− L0587 STRATAGENE ™ colon HT29 pBLUESCRIPT ™ (#937221) SK− L0588 STRATAGENE ™ endothelial pBLUESCRIPT ™ cell 937223 SK− L0589 STRATAGENE ™ fetal retina pBLUESCRIPT ™ 937202 SK− L0590 STRATAGENE ™ fibroblast pBLUESCRIPT ™ (#937212) SK− L0591 STRATAGENE ™ HeLa cell s3 pBLUESCRIPT ™ 937216 SK− L0592 STRATAGENE ™ hNT neuron pBLUESCRIPT ™ (#937233) SK− L0593 STRATAGENE ™ pBLUESCRIPT ™ neuroepithelium (#937231) SK− L0594 STRATAGENE ™ pBLUESCRIPT ™ neuroepithelium NT2RAMI SK− 937234 L0595 STRATAGENE ™ NT2 neuronal neuroepithelial cells brain pBLUESCRIPT ™ precursor 937230 SK− L0596 STRATAGENE ™ colon colon pBLUESCRIPT ™ (#937204) SK− L0597 STRATAGENE ™ corneal stroma cornea pBLUESCRIPT ™ (#937222) SK− L0598 Morton Fetal Cochlea cochlea ear pBLUESCRIPT ™ SK− L0599 STRATAGENE ™ lung lung pBLUESCRIPT ™ (#937210) SK− L0600 Weizmann Olfactory Epithelium olfactory epithelium nose pBLUESCRIPT ™ SK− L0601 STRATAGENE ™ pancreas pancreas pBLUESCRIPT ™ (#937208) SK− L0602 Pancreatic Islet pancreatic islet pancreas pBLUESCRIPT ™ SK− L0603 STRATAGENE ™ placenta placenta pBLUESCRIPT ™ (#937225) SK− L0604 STRATAGENE ™ muscle muscle skeletal pBLUESCRIPT ™ 937209 muscle SK− L0605 STRATAGENE ™ fetal spleen fetal spleen spleen pBLUESCRIPT ™ (#937205) SK− L0606 NCI_CGAP_Lym5 follicular lymphoma lymph node pBLUESCRIPT ™ SK− L0607 NCI_CGAP_Lym6 mantle cell lymphoma lymph node pBLUESCRIPT ™ SK− L0608 STRATAGENE ™ lung lung carcinoma lung NCI-H69 pBLUESCRIPT ™ carcinoma 937218 SK− L0609 Schiller astrocytoma astrocytoma brain pBLUESCRIPT ™ SK− (STRATAGENE ™) L0610 Schiller glioblastoma multiforme glioblastoma multiforme brain pBLUESCRIPT ™ SK− (STRATAGENE ™) L0611 Schiller meningioma meningioma brain pBLUESCRIPT ™ SK− (STRATAGENE ™) L0612 Schiller oligodendroglioma oligodendroglioma brain pBLUESCRIPT ™ SK− (STRATAGENE ™) L0615 22 week old human fetal liver pBLUESCRIPT ™ cDNA library II SK− L0616 Chromosome 21 exon pBLUESCRIPT ™ IIKS+ L0617 Chromosome 22 exon pBLUESCRIPT ™ IIKS+ L0618 Chromosome 9 exon pBLUESCRIPT ™ IIKS+ L0619 Chromosome 9 exon II pBLUESCRIPT ™ IIKS+ L0622 HM1 pcDNAII (Invitrogen) L0623 HM3 pectoral muscle (after pcDNAII mastectomy) (Invitrogen) L0625 NCI_CGAP_AR1 bulk alveolar tumor pCMV- SPORT2 L0626 NCI_CGAP_GC1 bulk germ cell seminoma pCMV- SPORT2 L0627 NCI_CGAP_Co1 bulk tumor colon pCMV- SPORT2 L0628 NCI_CGAP_Ov1 ovary bulk tumor ovary pCMV- SPORT2 L0629 NCI_CGAP_Mel3 metastatic melanoma to bowel (skin pCMV- bowel primary) SPORT4 L0630 NCI_CGAP_CNS1 substantia nigra brain pCMV- SPORT4 L0631 NCI_CGAP_Br7 breast pCMV- SPORT4 L0632 NCI_CGAP_Li5 hepatic adenoma liver pCMV- SPORT4 L0633 NCI_CGAP_Lu6 small cell carcinoma lung pCMV- SPORT4 L0634 NCI_CGAP_Ov8 serous adenocarcinoma ovary pCMV- SPORT4 L0635 NCI_CGAP_PNS1 dorsal root ganglion peripheral pCMV- nervous SPORT4 system L0636 NCI_CGAP_Pit1 four pooled pituitary brain pCMV- adenomas SPORT6 (LIFE TECHNOLOGIES ™) L0637 NCI_CGAP_Brn53 three pooled meningiomas brain pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L0638 NCI_CGAP_Brn35 tumor, 5 pooled (see brain pCMV- description) SPORT6 (LIFE TECHNOLOGIES ™) L0639 NCI_CGAP_Brn52 tumor, 5 pooled (see brain pCMV- description) SPORT6 (LIFE TECHNOLOGIES ™) L0640 NCI_CGAP_Br18 four pooled high-grade breast pCMV- tumors, including two SPORT6 (LIFE prima TECHNOLOGIES ™) L0641 NCI_CGAP_Co17 juvenile granulosa tumor colon pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L0642 NCI_CGAP_Co18 moderately differentiated colon pCMV- adenocarcinoma SPORT6 (LIFE TECHNOLOGIES ™) L0643 NCI_CGAP_Co19 moderately differentiated colon pCMV- adenocarcinoma SPORT6 (LIFE TECHNOLOGIES ™) L0644 NCI_CGAP_Co20 moderately differentiated colon pCMV- adenocarcinoma SPORT6 (LIFE TECHNOLOGIES ™) L0645 NCI_CGAP_Co21 moderately differentiated colon pCMV- adenocarcinoma SPORT6 (LIFE TECHNOLOGIES ™) L0646 NCI_CGAP_Co14 moderately-differentiated colon pCMV- adenocarcinoma SPORT6 (LIFE TECHNOLOGIES ™) L0647 NCI_CGAP_Sar4 five pooled sarcomas, connective pCMV- including myxoid tissue SPORT6 (LIFE liposarcoma TECHNOLOGIES ™) L0648 NCI_CGAP_Eso2 squamous cell carcinoma esophagus pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L0649 NCI_CGAP_GU1 2 pooled high-grade genitourinary pCMV- transitional cell tumors tract SPORT6 (LIFE TECHNOLOGIES ™) L0650 NCI_CGAP_Kid13 2 pooled Wilms'' tumors, kidney pCMV- one primary and one metast SPORT6 (LIFE TECHNOLOGIES ™) L0651 NCI_CGAP_Kid8 renal cell tumor kidney pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L0652 NCI_CGAP_Lu27 four pooled poorly- lung pCMV- differentiated SPORT6 (LIFE adenocarcinomas TECHNOLOGIES ™) L0653 NCI_CGAP_Lu28 two pooled squamous cell lung pCMV- carcinomas SPORT6 (LIFE TECHNOLOGIES ™) L0654 NCI_CGAP_Lu31 lung, cell line pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L0655 NCI_CGAP_Lym12 lymphoma, follicular mixed lymph node pCMV- small and large cell SPORT6 (LIFE TECHNOLOGIES ™) L0656 NCI_CGAP_Ov38 normal epithelium ovary pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L0657 NCI_CGAP_Ov23 tumor, 5 pooled (see ovary pCMV- description) SPORT6 (LIFE TECHNOLOGIES ™) L0658 NCI_CGAP_Ov35 tumor, 5 pooled (see ovary pCMV- description) SPORT6 (LIFE TECHNOLOGIES ™) L0659 NCI_CGAP_Pan1 adenocarcinoma pancreas pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L0661 NCI_CGAP_Mel15 malignant melanoma, skin pCMV- metastatic to lymph node SPORT6 (LIFE TECHNOLOGIES ™) L0662 NCI_CGAP_Gas4 poorly differentiated stomach pCMV- adenocarcinoma with signet r SPORT6 (LIFE TECHNOLOGIES ™) L0663 NCI_CGAP_Ut2 moderately-differentiated uterus pCMV- endometrial adenocarcino SPORT6 (LIFE TECHNOLOGIES ™) L0664 NCI_CGAP_Ut3 poorly-differentiated uterus pCMV- endometrial SPORT6 (LIFE adenocarcinoma, TECHNOLOGIES ™) L0665 NCI_CGAP_Ut4 serous papillary carcinoma, uterus pCMV- high grade, 2 pooled t SPORT6 (LIFE TECHNOLOGIES ™) L0666 NCI_CGAP_Ut1 well-differentiated uterus pCMV- endometrial SPORT6 (LIFE adenocarcinoma, 7 TECHNOLOGIES ™) L0667 NCI_CGAP_CML1 myeloid cells, 18 pooled whole blood pCMV- CML cases, BCR/ABL SPORT6 (LIFE rearra TECHNOLOGIES ™) L0669 Human MCF7 cDNA subtracted breast adenocarcinoma breast MCF7 pCR II with MDA-MB-231 cDNA [Invitrogen] L0681 Stanley Frontal SN individual frontal lobe (see brain pCR2.1 description) (Invitrogen) L0682 Stanley Frontal NB pool 2 frontal lobe (see brain pCR2.1-TOPO description) (Invitrogen) L0683 Stanley Frontal NS pool 2 frontal lobe (see brain pCR2.1-TOPO description) (Invitrogen) L0684 Stanley Frontal SB pool 1 frontal lobe (see brain pCR2.1-TOPO description) (Invitrogen) L0685 Stanley Frontal SN pool 1 frontal lobe (see brain pCR2.1-TOPO description) (Invitrogen) L0686 Stanley Frontal SN pool 2 frontal lobe (see brain pCR2.1-TOPO description) (Invitrogen) L0687 Stanley Hippocampus NB pool 1 hippocampus (see brain pCR2.1-TOPO description) (Invitrogen) L0688 Stanley Hippocampus SB pool 1 hippocampus (see brain pCR2.1-TOPO description) (Invitrogen) L0689 Stanley Hippocampus SN pool 1 hippocampus (see brain pCR2.1-TOPO description) (Invitrogen) L0690 Testis, Subtracted pCR II [Invitrogen] L0695 Human Glialblastoma Cell Brain BT-325 pCR II [Invitrogen] L0697 Testis 1 PGEM 5zf(+) L0698 Testis 2 PGEM 5zf(+) L0700 Outward Alu-primed hncDNA pGEM-3Z library L0708 NIH_MGC_17 rhabdomyosarcoma muscle pOTB7 L0709 NIH_MGC_21 choriocarcinoma placenta pOTB7 L0710 NIH_MGC_7 small cell carcinoma lung MGC3 pOTB7 L0716 PMA-induced HL60 cell PMA-induced pSport1 subtraction library HL60 human leukemic cell line L0717 Gessler Wilms tumor pSport1 L0718 Testis 5 pSport1 L0719 human embryo cDNA library Whole embryo pSport1 L0720 PN001-Normal Human Prostate prostate pSport1 L0731 Soares_pregnant_uterus_NbHPU uterus pT7T3-Pac L0738 Human colorectal cancer pT7T3D L0740 Soares melanocyte 2NbHM melanocyte pT7T3D (PHARMACIA ™) with a modified polylinker L0741 Soares adult brain N2b4HB55Y brain pT7T3D (PHARMACIA ™) with a modified polylinker L0742 Soares adult brain N2b5HB55Y brain pT7T3D (PHARMACIA ™) with a modified polylinker L0743 Soares breast 2NbHBst breast pT7T3D (PHARMACIA ™) with a modified polylinker L0744 Soares breast 3NbHBst breast pT7T3D (PHARMACIA ™) with a modified polylinker L0745 Soares retina N2b4HR retina eye pT7T3D (PHARMACIA ™) with a modified polylinker L0746 Soares retina N2b5HR retina eye pT7T3D (PHARMACIA ™) with a modified polylinker L0747 Soares_fetal_heart_NbHH19W heart pT7T3D (PHARMACIA ™) with a modified polylinker L0748 Soares fetal liver spleen 1NFLS Liver and pT7T3D Spleen (PHARMACIA ™) with a modified polylinker L0749 Soares_fetal_liver_spleen_(—) Liver and pT7T3D 1NFLS_S1 Spleen (PHARMACIA ™) with a modified polylinker L0750 Soares_fetal_lung_NbHL19W lung pT7T3D (PHARMACIA ™) with a modified polylinker L0751 Soares ovary tumor NbHOT ovarian tumor ovary pT7T3D (PHARMACIA ™) with a modified polylinker L0752 Soares_parathyroid_tumor_NbHPA parathyroid tumor parathyroid pT7T3D gland (PHARMACIA ™) with a modified polylinker L0753 Soares_pineal_gland_N3HPG pineal gland pT7T3D (PHARMACIA ™) with a modified polylinker L0754 Soares placenta Nb2HP placenta pT7T3D (PHARMACIA ™) with a modified polylinker L0755 Soares_placenta_8to9weeks_(—) placenta pT7T3D 2NbHP8to9W (PHARMACIA ™) with a modified polylinker L0756 Soares_multiple_sclerosis_(—) multiple sclerosis lesions pT7T3D 2NbHMSP (PHARMACIA ™) with a modified polylinker V_TYPE L0757 Soares_senescent_fibroblasts_(—) senescent fibroblast pT7T3D NbHSF (PHARMACIA ™) with a modified polylinker V_TYPE L0758 Soares_testis_NHT pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0759 Soares_total_fetus_Nb2HF8_9w pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0760 Barstead aorta HPLRB3 aorta pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0761 NCI_CGAP_CLL1 B-cell, chronic lymphotic pT7T3D-Pac leukemia (PHARMACIA ™) with a modified polylinker L0762 NCI_CGAP_Br1.1 breast pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0763 NCI_CGAP_Br2 breast pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0764 NCI_CGAP_Co3 colon pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0765 NCI_CGAP_Co4 colon pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0766 NCI_CGAP_GCB1 germinal center B cell pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0767 NCI_CGAP_GC3 pooled germ cell tumors pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0768 NCI_CGAP_GC4 pooled germ cell tumors pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0769 NCI_CGAP_Brn25 anaplastic brain pT7T3D-Pac oligodendroglioma (PHARMACIA ™) with a modified polylinker L0770 NCI_CGAP_Brn23 glioblastoma (pooled) brain pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0771 NCI_CGAP_Co8 adenocarcinoma colon pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0772 NCI_CGAP_Co10 colon tumor RER+ colon pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0773 NCI_CGAP_Co9 colon tumor RER+ colon pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0774 NCI_CGAP_Kid3 kidney pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0775 NCI_CGAP_Kid5 2 pooled tumors (clear cell kidney pT7T3D-Pac type) (PHARMACIA ™) with a modified polylinker L0776 NCI_CGAP_Lu5 carcinoid lung pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0777 Soares_NhHMPu_S1 Pooled human melanocyte, mixed (see pT7T3D-Pac fetal heart, and pregnant below) (PHARMACIA ™) with a modified polylinker L0778 Barstead pancreas HPLRB1 pancreas pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0779 Soares_NFL_T_GBC_S1 pooled pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0780 Soares_NSF_F8_9W_OT_PA_(—) pooled pT7T3D-Pac P_S1 (PHARMACIA ™) with a modified polylinker L0782 NCI_CGAP_Pr21 normal prostate prostate pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0783 NCI_CGAP_Pr22 normal prostate prostate pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0784 NCI_CGAP_Lei2 leiomyosarcoma soft tissue pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0785 Barstead spleen HPLRB2 spleen pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0786 Soares_NbHFB whole brain pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0787 NCI_CGAP_Sub1 pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0788 NCI_CGAP_Sub2 pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0789 NCI_CGAP_Sub3 pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0790 NCI_CGAP_Sub4 pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0791 NCI_CGAP_Sub5 pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0792 NCI_CGAP_Sub6 pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0793 NCI_CGAP_Sub7 pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0794 NCI_CGAP_GC6 pooled germ cell tumors pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0796 NCI_CGAP_Brn50 medulloblastoma brain pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0800 NCI_CGAP_Co16 colon tumor, RER+ colon pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0803 NCI_CGAP_Kid11 kidney pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0804 NCI_CGAP_Kid12 2 pooled tumors (clear cell kidney pT7T3D-Pac type) (PHARMACIA ™) with a modified polylinker L0805 NCI_CGAP_Lu24 carcinoid lung pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0806 NCI_CGAP_Lu19 squamous cell carcinoma, lung pT7T3D-Pac poorly differentiated (4 (PHARMACIA ™) with a modified polylinker L0807 NCI_CGAP_Ov18 fibrotheoma ovary pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0808 Barstead prostate BPH HPLRB41 prostate pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0809 NCI_CGAP_Pr28 prostate pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L0811 BATM2 PTZ18 L0879 BT0254 breast puc18 L0930 BT0314 breast puc18 L0946 BT0333 breast puc18 L0988 BT0387 breast puc18 L1057 BT0559 breast puc18 L1278 BN0005 breast_normal puc18 L1430 CT0225 colon puc18 L1441 CT0249 colon puc18 L1446 CT0254 colon puc18 L1477 CT0297 colon puc18 L1499 CT0322 colon puc18 L1548 CN0007 colon_normal puc18 L1561 CN0026 colon_normal puc18 L1562 CN0027 colon_normal puc18 L1607 DT0041 denis_drash puc18 L1651 HT0059 head_neck puc18 L1727 HT0158 head_neck puc18 L1788 HT0229 head_neck puc18 L1819 HT0268 head_neck puc18 L1872 HT0335 head_neck puc18 L1877 HT0340 head_neck puc18 L1878 HT0342 head_neck puc18 L1886 HT0350 head_neck puc18 L1894 HT0366 head_neck puc18 L1942 HT0452 head_neck puc18 L1948 HT0470 head_neck puc18 L2094 ST0125 stomach puc18 L2138 ST0186 stomach puc18 L2174 ST0240 stomach puc18 L2197 ST0278 stomach puc18 L2210 ST0293 stomach puc18 L2242 subtracted 3″ EST library pancreas AsPC- pUC18 1(ATCC ™:CRL- 1682) L2245 NEM subtracted human fetal pUEX1 kidney cDNA L2250 Human cerebral cortex cerebral cortex L2251 Human fetal lung Fetal lung L2252 Human placenta placenta L2255 GLC corresponding non pBLUESCRIPT ™ cancerous liver tissue SK− L2257 NIH_MGC_65 adenocarcinoma colon pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L2258 NIH_MGC_67 retinoblastoma eye pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L2259 NIH_MGC_68 large cell carcinoma lung pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L2260 NIH_MGC_69 large cell carcinoma, lung pCMV- undifferentiated SPORT6 (LIFE TECHNOLOGIES ™) L2261 NIH_MGC_70 epithelioid carcinoma pancreas pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L2262 NIH_MGC_72 melanotic melanoma skin pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L2263 NIH_MGC_66 adenocarcinoma ovary pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L2264 NIH_MGC_71 leiomyosarcoma uterus pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L2265 NIH_MGC_39 adenocarcinoma pancreas pOTB7 L2269 NCI_CGAP_Thy11 follicular carcinoma thyroid pAMP10 L2270 Lupski_dorsal_root_ganglion dorsal root ganglia pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L2277 BT0626 breast puc18 L2279 BT0659 breast puc18 L2281 BT0701 breast puc18 L2283 BT0705 breast puc18 L2285 BT0723 breast puc18 L2289 BT0757 breast puc18 L2291 BT0760 breast puc18 L2293 BT0762 breast puc18 L2294 BT0763 breast puc18 L2300 BT0789 breast puc18 L2301 BT0792 breast puc18 L2308 CT0383 colon puc18 L2317 CT0400 colon puc18 L2323 CT0406 colon puc18 L2328 CT0412 colon puc18 L2332 CT0416 colon puc18 L2333 CT0417 colon puc18 L2336 CT0428 colon puc18 L2338 CT0432 colon puc18 L2339 CT0434 colon puc18 L2346 CT0483 colon puc18 L2348 CT0491 colon puc18 L2352 UT0001 uterus_tumor puc18 L2353 UT0003 uterus_tumor puc18 L2354 UT0005 uterus_tumor puc18 L2357 UT0021 uterus_tumor puc18 L2359 UT0023 uterus_tumor puc18 L2361 UT0028 uterus_tumor puc18 L2364 UT0033 uterus_tumor puc18 L2367 UT0039 uterus_tumor puc18 L2368 UT0041 uterus_tumor puc18 L2372 NN0034 nervous_normal puc18 L2377 NN0054 nervous_normal puc18 L2380 NN0068 nervous_normal puc18 L2381 NN0070 nervous_normal puc18 L2382 NN0073 nervous_normal puc18 L2389 NN0087 nervous_normal puc18 L2400 NN0116 nervous_normal puc18 L2402 NN0118 nervous_normal puc18 L2412 NN0136 nervous_normal puc18 L2413 NN0141 nervous_normal puc18 L2439 NN1022 nervous_normal puc18 L2440 NN1023 nervous_normal puc18 L2449 NN1063 nervous_normal puc18 L2450 NN1065 nervous_normal puc18 L2462 NN1089 nervous_normal puc18 L2464 NN1104 nervous_normal puc18 L2466 NN1111 nervous_normal puc18 L2467 NN1112 nervous_normal puc18 L2471 NN1123 nervous_normal puc18 L2472 NN1124 nervous_normal puc18 L2477 HT0408 head_neck puc18 L2478 HT0445 head_neck puc18 L2482 HT0497 head_neck puc18 L2486 HT0527 head_neck puc18 L2487 HT0542 head_neck puc18 L2490 HT0545 head_neck puc18 L2491 HT0559 head_neck puc18 L2493 HT0576 head_neck puc18 L2494 HT0577 head_neck puc18 L2495 HT0594 head_neck puc18 L2497 HT0618 head_neck puc18 L2498 HT0619 head_neck puc18 L2499 HT0622 head_neck puc18 L2500 HT0623 head_neck puc18 L2504 HT0636 head_neck puc18 L2506 HT0638 head_neck puc18 L2513 HT0678 head_neck puc18 L2518 HT0697 head_neck puc18 L2519 HT0698 head_neck puc18 L2521 HT0702 head_neck puc18 L2522 HT0704 head_neck puc18 L2525 HT0710 head_neck puc18 L2528 HT0713 head_neck puc18 L2535 HT0723 head_neck puc18 L2539 HT0727 head_neck puc18 L2540 HT0728 head_neck puc18 L2543 HT0734 head_neck puc18 L2545 HT0736 head_neck puc18 L2550 HT0743 head_neck puc18 L2551 HT0744 head_neck puc18 L2552 HT0745 head_neck puc18 L2558 HT0756 head_neck puc18 L2560 HT0758 head_neck puc18 L2562 HT0760 head_neck puc18 L2565 HT0764 head_neck puc18 L2570 HT0771 head_neck puc18 L2571 HT0773 head_neck puc18 L2578 HT0785 head_neck puc18 L2581 HT0790 head_neck puc18 L2587 HT0797 head_neck puc18 L2596 HT0807 head_neck puc18 L2598 HT0809 head_neck puc18 L2599 HT0810 head_neck puc18 L2610 HT0837 head_neck puc18 L2615 HT0843 head_neck puc18 L2618 HT0847 head_neck puc18 L2630 HT0865 head_neck puc18 L2634 HT0872 head_neck puc18 L2635 HT0875 head_neck puc18 L2637 HT0877 head_neck puc18 L2638 HT0878 head_neck puc18 L2640 HT0881 head_neck puc18 L2644 HT0886 head_neck puc18 L2647 HT0894 head_neck puc18 L2649 HT0905 head_neck puc18 L2650 HT0934 head_neck puc18 L2651 NIH_MGC_20 melanotic melanoma skin pOTB7 L2652 NIH_MGC_57 glioblastoma brain pDNR-LIB (CLONTECH ™) L2653 NIH_MGC_58 hypernephroma kidney pDNR-LIB (CLONTECH ™) L2654 NIH_MGC_9 adenocarcinoma cell line ovary pOTB7 L2655 NIH_MGC_55 from acute myelogenous bone marrow pDNR-LIB leukemia (CLONTECH ™) L2657 NIH_MGC_54 from chronic myelogenous bone marrow pDNR-LIB leukemia (CLONTECH ™) L2667 NT0013 nervous_tumor puc18 L2668 NT0018 nervous_tumor puc18 L2669 NT0022 nervous_tumor puc18 L2670 NT0023 nervous_tumor puc18 L2671 NT0024 nervous_tumor puc18 L2673 NT0028 nervous_tumor puc18 L2674 NT0029 nervous_tumor puc18 L2675 NT0033 nervous_tumor puc18 L2677 NT0039 nervous_tumor puc18 L2681 NT0048 nervous_tumor puc18 L2683 NT0053 nervous_tumor puc18 L2686 NT0058 nervous_tumor puc18 L2689 NT0073 nervous_tumor puc18 L2696 NT0084 nervous_tumor puc18 L2702 NT0098 nervous_tumor puc18 L2705 NT0101 nervous_tumor puc18 L2706 NT0102 nervous_tumor puc18 L2708 NT0104 nervous_tumor puc18 L2709 NT0105 nervous_tumor puc18 L2716 NT0117 nervous_tumor puc18 L2730 GN0021 placenta_normal puc18 L2731 GN0023 placenta_normal puc18 L2733 GN0037 placenta_normal puc18 L2737 GN0047 placenta_normal puc18 L2738 GN0049 placenta_normal puc18 L2744 FT0004 prostate_tumor puc18 L2754 FT0022 prostate_tumor puc18 L2755 FT0023 prostate_tumor puc18 L2756 FT0024 prostate_tumor puc18 L2757 FT0025 prostate_tumor puc18 L2758 FT0027 prostate_tumor puc18 L2759 FT0028 prostate_tumor puc18 L2763 FT0039 prostate_tumor puc18 L2766 FT0042 prostate_tumor puc18 L2767 FT0044 prostate_tumor puc18 L2771 FT0050 prostate_tumor puc18 L2777 FT0056 prostate_tumor puc18 L2779 FT0058 prostate_tumor puc18 L2788 FT0071 prostate_tumor puc18 L2791 FT0077 prostate_tumor puc18 L2793 FT0080 prostate_tumor puc18 L2799 FT0096 prostate_tumor puc18 L2800 FT0097 prostate_tumor puc18 L2804 FT0103 prostate_tumor puc18 L2809 FT0117 prostate_tumor puc18 L2810 FT0119 prostate_tumor puc18 L2811 FT0122 prostate_tumor puc18 L2812 FT0123 prostate_tumor puc18 L2814 FT0128 prostate_tumor puc18 L2815 FT0129 prostate_tumor puc18 L2817 FT0131 prostate_tumor puc18 L2819 FT0134 prostate_tumor puc18 L2831 FT0162 prostate_tumor puc18 L2833 FT0164 prostate_tumor puc18 L2836 FT0169 prostate_tumor puc18 L2842 UM0009 uterus puc18 L2843 UM0017 uterus puc18 L2844 UM0018 uterus puc18 L2845 UM0021 uterus puc18 L2846 UM0022 uterus puc18 L2848 UM0053 uterus puc18 L2852 UM0077 uterus puc18 L2854 UM0091 uterus puc18 L2865 AN0004 amnion_normal puc18 L2869 AN0012 amnion_normal puc18 L2870 AN0013 amnion_normal puc18 L2877 AN0027 amnion_normal puc18 L2878 AN0029 amnion_normal puc18 L2879 AN0032 amnion_normal puc18 L2884 AN0041 amnion_normal puc18 L2888 AN0056 amnion_normal puc18 L2893 AN0062 amnion_normal puc18 L2899 AN0094 amnion_normal puc18 L2902 BN0036 breast_normal puc18 L2903 BN0039 breast_normal puc18 L2904 BN0042 breast_normal puc18 L2905 BN0046 breast_normal puc18 L2906 BN0047 breast_normal puc18 L2909 BN0067 breast_normal puc18 L2910 BN0070 breast_normal puc18 L2914 BN0090 breast_normal puc18 L2915 BN0098 breast_normal puc18 L2918 BN0114 breast_normal puc18 L2919 BN0115 breast_normal puc18 L2924 BN0138 breast_normal puc18 L2938 BN0174 breast_normal puc18 L2962 BN0221 breast_normal puc18 L2978 BN0247 breast_normal puc18 L2985 BN0257 breast_normal puc18 L2987 BN0259 breast_normal puc18 L2991 BN0264 breast_normal puc18 L2999 BN0273 breast_normal puc18 L3001 BN0275 breast_normal puc18 L3002 BN0276 breast_normal puc18 L3010 BN0294 breast_normal puc18 L3011 BN0295 breast_normal puc18 L3012 BN0296 breast_normal puc18 L3019 BN0303 breast_normal puc18 L3020 BN0304 breast_normal puc18 L3041 BN0332 breast_normal puc18 L3058 EN0004 lung_normal puc18 L3066 EN0018 lung_normal puc18 L3071 EN0026 lung_normal puc18 L3078 EN0042 lung_normal puc18 L3080 ET0001 lung_tumor puc18 L3081 ET0005 lung_tumor puc18 L3089 ET0018 lung_tumor puc18 L3092 ET0023 lung_tumor puc18 L3093 ET0024 lung_tumor puc18 L3095 ET0027 lung_tumor puc18 L3104 ET0041 lung_tumor puc18 L3109 ET0046 lung_tumor puc18 L3111 ET0058 lung_tumor puc18 L3117 ET0068 lung_tumor puc18 L3118 ET0070 lung_tumor puc18 L3119 ET0072 lung_tumor puc18 L3127 ET0084 lung_tumor puc18 L3128 MT0016 marrow puc18 L3132 MT0022 marrow puc18 L3134 MT0024 marrow puc18 L3140 MT0031 marrow puc18 L3144 MT0035 marrow puc18 L3153 MT0049 marrow puc18 L3154 MT0050 marrow puc18 L3158 MT0057 marrow puc18 L3160 MT0059 marrow puc18 L3180 MT0101 marrow puc18 L3181 MT0107 marrow puc18 L3182 MT0108 marrow puc18 L3184 MT0111 marrow puc18 L3186 MT0113 marrow puc18 L3199 OT0019 ovary puc18 L3204 OT0034 ovary puc18 L3207 OT0063 ovary puc18 L3210 OT0067 ovary puc18 L3211 OT0072 ovary puc18 L3212 OT0076 ovary puc18 L3213 OT0078 ovary puc18 L3215 OT0083 ovary puc18 L3216 OT0086 ovary puc18 L3217 OT0091 ovary puc18 L3226 FN0019 prostate_normal puc18 L3250 FN0058 prostate_normal puc18 L3255 FN0064 prostate_normal puc18 L3262 FN0073 prostate_normal puc18 L3264 FN0080 prostate_normal puc18 L3271 FN0094 prostate_normal puc18 L3274 FN0098 prostate_normal puc18 L3278 FN0104 prostate_normal puc18 L3280 FN0106 prostate_normal puc18 L3281 FN0107 prostate_normal puc18 L3295 FN0138 prostate_normal puc18 L3297 FN0140 prostate_normal puc18 L3300 FN0143 prostate_normal puc18 L3311 FN0180 prostate_normal puc18 L3312 FN0181 prostate_normal puc18 L3316 FN0188 prostate_normal puc18 L3327 SN0024 stomach_normal puc18 L3330 SN0041 stomach_normal puc18 L3336 SN0066 stomach_normal puc18 L3352 TN0027 testis_normal puc18 L3355 TN0032 testis_normal puc18 L3357 TN0034 testis_normal puc18 L3358 TN0035 testis_normal puc18 L3359 TN0036 testis_normal puc18 L3369 TN0065 testis_normal puc18 L3372 TN0068 testis_normal puc18 L3374 TN0070 testis_normal puc18 L3377 TN0079 testis_normal puc18 L3378 TN0080 testis_normal puc18 L3385 Homo sapiens HeLa HeLa L3387 GKB hepatocellular carcinoma pBLUESCRIPT ™ SK− L3388 GKC hepatocellular carcinoma pBLUESCRIPT ™ SK− L3389 GKD hepatocellular carcinoma pBLUESCRIPT ™ SK− L3391 NIH_MGC_53 carcinoma, cell line bladder pDNR-LIB (CLONTECH ™) L3401 AN0085 amnion_normal puc18 L3402 AN0086 amnion_normal puc18 L3403 AN0087 amnion_normal puc18 L3404 AN0089 amnion_normal puc18 L3421 BT0634 breast puc18 L3431 CT0451 colon puc18 L3432 CT0461 colon puc18 L3435 CT0465 colon puc18 L3443 CT0482 colon puc18 L3445 CT0497 colon puc18 L3450 CT0508 colon puc18 L3459 FT0175 prostate_tumor puc18 L3463 GN0016 placenta_normal puc18 L3464 GN0018 placenta_normal puc18 L3466 GN0020 placenta_normal puc18 L3476 GN0051 placenta_normal puc18 L3480 GN0057 placenta_normal puc18 L3484 GN0067 placenta_normal puc18 L3485 GN0070 placenta_normal puc18 L3490 GN0075 placenta_normal puc18 L3491 GN0076 placenta_normal puc18 L3494 HT0539 head_neck puc18 L3495 HT0570 head_neck puc18 L3496 HT0572 head_neck puc18 L3497 HT0600 head_neck puc18 L3499 HT0617 head_neck puc18 L3503 HT0870 head_neck puc18 L3504 HT0873 head_neck puc18 L3506 HT0879 head_neck puc18 L3511 HT0900 head_neck puc18 L3516 HT0913 head_neck puc18 L3518 HT0915 head_neck puc18 L3520 HT0918 head_neck puc18 L3521 HT0919 head_neck puc18 L3526 HT0931 head_neck puc18 L3530 HT0939 head_neck puc18 L3540 MT0126 marrow puc18 L3546 NN0044 nervous_normal puc18 L3554 OT0035 ovary puc18 L3560 TN0023 testis_normal puc18 L3561 TN0025 testis_normal puc18 L3562 TN0030 testis_normal puc18 L3563 TN0037 testis_normal puc18 L3565 TN0045 testis_normal puc18 L3566 TN0046 testis_normal puc18 L3576 TN0086 testis_normal puc18 L3585 TN0119 testis_normal puc18 L3586 TN0120 testis_normal puc18 L3592 TN0129 testis_normal puc18 L3603 UM0093 uterus puc18 L3605 UM0104 uterus puc18 L3609 UT0007 uterus_tumor puc18 L3612 UT0011 uterus_tumor puc18 L3618 UT0050 uterus_tumor puc18 L3625 UT0063 uterus_tumor puc18 L3630 UT0071 uterus_tumor puc18 L3631 UT0072 uterus_tumor puc18 L3632 UT0074 uterus_tumor puc18 L3634 NIH_MGC_56 primitive neuroectoderm brain pDNR-LIB (CLONTECH ™) L3635 NIH_MGC_62 melanotic melanoma, high skin pDNR-LIB MDR (CLONTECH ™) L3636 NIH_MGC_73 brain pDNR-LIB (CLONTECH ™) L3637 NIH_MGC_74 heart pDNR-LIB (CLONTECH ™) L3638 NIH_MGC_78 pancreas pDNR-LIB (CLONTECH ™) L3641 NIH_MGC_83 prostate pDNR-LIB (CLONTECH ™) L3642 ADA Adrenal gland pBLUESCRIPT ™ SK− L3643 ADB Adrenal gland pBLUESCRIPT ™ SK− L3644 ADC Adrenal gland pBLUESCRIPT ™ SK− L3645 Cu adrenal cortico adenoma for pBLUESCRIPT ™ Cushing''s syndrome SK− L3646 DCA pTriplEx2 L3647 Human HO-1 melanoma cells L3649 DCB pTriplEx2 L3651 FHTA hypothalamus pTriplEx2 L3652 FHTB hypothalamus pTriplEx2 L3653 HTB Hypothalamus pBLUESCRIPT ™ SK− L3655 HTC Hypothalamus pBLUESCRIPT ™ SK− L3656 HTE Hypothalamus pBLUESCRIPT ™ SK− L3657 HTF Hypothalamus pBLUESCRIPT ™ SK− L3658 cdA pheochromocytoma pTriplEx2 L3659 CB cord blood pBLUESCRIPT ™ L3660 NP1 pituitary pBLUESCRIPT ™ SK− L3661 NPA pituitary pBLUESCRIPT ™ SK− L3663 NIH_MGC_60 adenocarcinoma prostate pDNR-LIB (CLONTECH ™) L3664 NIH_MGC_61 embryonal carcinoma testis pDNR-LIB (CLONTECH ™) L3665 NIH_MGC_75 kidney pDNR-LIB (CLONTECH ™) L3666 NIH_MGC_77 lung pDNR-LIB (CLONTECH ™) L3667 NIH_MGC_79 placenta pDNR-LIB (CLONTECH ™) L3668 AN0063 amnion_normal puc18 L3672 AN0083 amnion_normal puc18 L3673 AN0084 amnion_normal puc18 L3684 BT0812 breast puc18 L3693 CI0018 colon_ins puc18 L3697 CS0012 colon_est puc18 L3699 CT0437 colon puc18 L3705 CT0486 colon puc18 L3709 CT0515 colon puc18 L3713 CT0524 colon puc18 L3718 CT0532 colon puc18 L3722 GN0030 placenta_normal puc18 L3724 GN0034 placenta_normal puc18 L3726 GN0038 placenta_normal puc18 L3728 GN0077 placenta_normal puc18 L3729 GN0079 placenta_normal puc18 L3738 GN0092 placenta_normal puc18 L3739 HT0540 head_neck puc18 L3744 HT0916 head_neck puc18 L3750 HT0945 head_neck puc18 L3752 HT0947 head_neck puc18 L3761 NN1141 nervous_normal puc18 L3763 SN0036 stomach_normal puc18 L3768 TN0073 testis_normal puc18 L3778 TN0112 testis_normal puc18 L3783 TN0136 testis_normal puc18 L3790 TN0150 testis_normal puc18 L3796 UT0042 uterus_tumor puc18 L3802 UT0052 uterus_tumor puc18 L3804 UT0073 uterus_tumor puc18 L3805 UT0075 uterus_tumor puc18 L3807 UT0077 uterus_tumor puc18 L3808 UT0078 uterus_tumor puc18 L3811 NPC pituitary pBLUESCRIPT ™ SK− L3812 NPD pituitary pBLUESCRIPT ™ SK− L3813 TP pituitary tumor pTriplEx2 L3814 BM Bone marrow pTriplEx2 L3815 MDS Bone marrow pTriplEx2 L3816 HEMBA1 whole embryo, mainly head pME18SFL3 L3817 HEMBB1 whole embryo, mainly body pME18SFL3 L3818 MAMMA1 mammary gland pME18SFL3 L3819 NIH_MGC_76 liver pDNR-LIB (CLONTECH ™) L3820 NIH_MGC_46 leiomyosarcoma cell line uterus pOTB7 L3821 NIH_MGC_48 primary B-cells from B-cells pOTB7 tonsils (cell line) L3822 NIH_MGC_59 mucoepidermoid carcinoma lung pDNR-LIB (CLONTECH ™) L3823 NT2RM1 NT2 pUC19FL3 L3824 NT2RM2 NT2 pME18SFL3 L3825 NT2RM4 NT2 pME18SFL3 L3826 NT2RP1 NT2 pUC19FL3 L3827 NT2RP2 NT2 pME18SFL3 L3828 NT2RP3 NT2 pME18SFL3 L3829 NT2RP4 NT2 pME18SFL3 L3831 OVARC1 ovary, tumor tissue pME18SFL3 L3832 PLACE1 placenta pME18SFL3 L3833 PLACE2 placenta pME18SFL3 L3834 PLACE3 placenta pME18SFL3 L3835 PLACE4 placenta pME18SFL3 L3836 SKNMC1 SK-N-MC pME18SFL3 L3837 THYRO1 thyroid gland pME18SFL3 L3839 Y79AA1 Y79 pME18SFL3 L3841 NIH_MGC_18 large cell carcinoma lung pOTB7 L3854 BT0817 breast puc18 L3871 NIH_MGC_19 neuroblastoma brain pOTB7 L3872 NCI_CGAP_Skn1 skin, normal, pCMV- 4 pooled sa SPORT6 (LIFE TECHNOLOGIES ™) L3873 Human esophageal carcinoma esophageal squamous cell pGEM-T mRNA carcinoma (Promega) L3904 NCI_CGAP_Brn64 glioblastoma with EGFR brain pCMV- amplification SPORT6 (LIFE TECHNOLOGIES ™) L3905 NCI_CGAP_Brn67 anaplastic brain pCMV- oligodendroglioma with SPORT6 (LIFE 1p/19q loss TECHNOLOGIES ™) L4497 NCI_CGAP_Br22 invasive ductal carcinoma, breast pCMV- 3 pooled samples SPORT6 (LIFE TECHNOLOGIES ™) L4500 NCI_CGAP_HN16 moderate to poorly mouth pAMP10 differentiated invasive carcino L4501 NCI_CGAP_Sub8 pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L4507 NCI_CGAP_Thy6 normal epithelium thyroid pAMP10 L4508 NCI_CGAP_Thy8 normal epithelium thyroid pAMP10 L4535 NCI_CGAP_Thy4 normal epithelium thyroid pAMP10 L4537 NCI_CGAP_Thy7 follicular adenoma (benign thyroid pAMP10 lesion) L4556 NCI_CGAP_HN13 squamous cell carcinoma tongue pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L4557 NCI_CGAP_Adr1 neuroblastoma adrenal gland pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L4558 NCI_CGAP_Pan3 pancreas pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L4559 NCI_CGAP_Thy3 follicular carcinoma thyroid pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L4560 NCI_CGAP_Ut7 tumor uterus pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L4669 NCI_CGAP_Ov41 serous papillary tumor ovary pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L4747 NCI_CGAP_Brn41 oligodendroglioma brain pT7T3D-Pac (PHARMACIA ™) with a modified polylinker L4753 NCI_CGAP_HN15 leukoplakia of the buccal mouth pAMP10 mucosa L4775 NCI_CGAP_Thy12 papillary carcinoma thyroid pAMP10 L5286 NCI_CGAP_Thy10 medullary carcinoma thyroid pAMP10 L5564 NCI_CGAP_HN20 normal pAMP1 head/neck tissue L5565 NCI_CGAP_Brn66 glioblastoma with probably brain pCMV- TP53 mutation and witho SPORT6 (LIFE TECHNOLOGIES ™) L5566 NCI_CGAP_Brn70 anaplastic brain pCMV- oligodendroglioma SPORT6.ccdb L5568 NCI_CGAP_HN21 nasopharyngeal carcinoma head/neck pAMP1 L5569 NCI_CGAP_HN17 normal epithelium nasopharynx pAMP10 L5570 NCI_CGAP_Co28 normal colonic mucosa colon pAMP1 L5572 NCI_CGAP_Co27 adenocarcinoma (mucinous colon pAMP1 component) L5574 NCI_CGAP_HN19 normal epithelium nasopharynx pAMP10 L5575 NCI_CGAP_Brn65 glioblastoma without EGFR brain pCMV- amplification SPORT6 (LIFE TECHNOLOGIES ™) L5622 NCI_CGAP_Skn3 skin pCMV- SPORT6 (LIFE TECHNOLOGIES ™) L5623 NCI_CGAP_Skn4 squamous cell carcinoma skin pCMV- SPORT6 (LIFE TECHNOLOGIES ™) N0003 Human Fetal Brain Human Fetal Brain N0004 Human Hippocampus Human Hippocampus N0006 Human Fetal Brain Human Fetal Brain N0007 Human Hippocampus Human Hippocampus N0009 Human Hippocampus, Human Hippocampus prescreened N0011 Human Brain Human Brain S0001 Brain frontal cortex Brain frontal cortex Brain LAMBDA ZAP ™ II S0002 Monocyte activated Monocyte-activated blood Cell Line UNI-ZAP ™ XR S0003 Human Osteoclastoma Osteoclastoma bone disease UNI-ZAP ™ XR S0004 Prostate Prostate BPH Prostate LAMBDA ZAP ™ II S0005 Heart Heart-left ventricle Heart pCDNA S0006 Neuroblastoma Human Neural Blastoma disease pCDNA S0007 Early Stage Human Brain Human Fetal Brain UNI-ZAP ™ XR S0008 Osteoclastoma Osteoclastoma bone disease UNI-ZAP ™ XR S0010 Human Amygdala Amygdala UNI-ZAP ™ XR S0011 STROMAL- Osteoclastoma bone disease UNI-ZAP ™ OSTEOCLASTOMA XR S0013 Prostate Prostate prostate UNI-ZAP ™ XR S0014 Kidney Cortex Kidney cortex Kidney UNI-ZAP ™ XR S0015 Kidney medulla Kidney medulla Kidney UNI-ZAP ™ XR S0016 Kidney Pyramids Kidney pyramids Kidney UNI-ZAP ™ XR S0020 Seven Trans Membrane Receptor 7TMD1 Family S0021 Whole brain Whole brain Brain ZAP EXPRESS ™ S0022 Human Osteoclastoma Stromal Osteoclastoma Stromal UNI-ZAP ™ Cells - unamplified Cells XR S0023 Human Kidney Cortex - Human Kidney Cortex unamplified S0024 Human Kidney Medulla - Human Kidney Medulla unamplified S0025 Human Kidney Pyramids - Human Kidney Pyramids unamplified S0026 Stromal cell TF274 stromal cell Bone marrow Cell Line UNI-ZAP ™ XR S0027 Smooth muscle, serum treated Smooth muscle Pulmanary Cell Line UNI-ZAP ™ artery XR S0028 Smooth muscle, control Smooth muscle Pulmanary Cell Line UNI-ZAP ™ artery XR S0029 brain stem Brain stem brain UNI-ZAP ™ XR S0030 Brain pons Brain Pons Brain UNI-ZAP ™ XR S0031 Spinal cord Spinal cord spinal cord UNI-ZAP ™ XR S0032 Smooth muscle-ILb induced Smooth muscle Pulmanary Cell Line UNI-ZAP ™ artery XR S0035 Brain medulla oblongata Brain medulla oblongata Brain UNI-ZAP ™ XR S0036 Human Substantia Nigra Human Substantia Nigra UNI-ZAP ™ XR S0037 Smooth muscle, IL1b induced Smooth muscle Pulmanary Cell Line UNI-ZAP ™ artery XR S0038 Human Whole Brain #2 - Oligo Human Whole Brain #2 ZAP dT >1.5 Kb EXPRESS ™ S0039 Hypothalamus Hypothalamus Brain UNI-ZAP ™ XR S0040 Adipocytes Human Adipocytes from UNI-ZAP ™ Osteoclastoma XR S0041 Thalamus Human Thalamus UNI-ZAP ™ XR S0042 Testes Human Testes ZAP EXPRESS ™ S0044 Prostate BPH prostate BPH Prostate disease UNI-ZAP ™ XR S0045 Endothelial cells-control Endothelial cell endothelial Cell Line UNI-ZAP ™ cell-lung XR S0046 Endothelial-induced Endothelial cell endothelial Cell Line UNI-ZAP ™ cell-lung XR S0048 Human Hypothalamus, Human Hypothalamus, disease UNI-ZAP ™ Alzheimer''s Alzheimer''s XR S0049 Human Brain, Striatum Human Brain, Striatum UNI-ZAP ™ XR S0050 Human Frontal Cortex, Human Frontal Cortex, disease UNI-ZAP ™ Schizophrenia Schizophrenia XR S0051 Human Human Hypothalamus, disease UNI-ZAP ™ Hypothalmus, Schizophrenia Schizophrenia XR S0052 neutrophils control human neutrophils blood Cell Line UNI-ZAP ™ XR S0053 Neutrophils IL-1 and LPS human neutrophil induced blood Cell Line UNI-ZAP ™ induced XR S0106 STRIATUM DEPRESSION BRAIN disease UNI-ZAP ™ XR S0110 Brain Amygdala Depression Brain disease UNI-ZAP ™ XR S0112 Hypothalamus Brain UNI-ZAP ™ XR S0114 Anergic T-cell Anergic T-cell Cell Line UNI-ZAP ™ XR S0116 Bone marrow Bone marrow Bone marrow UNI-ZAP ™ XR S0118 Smooth muscle control 2 Smooth muscle Pulmanary Cell Line UNI-ZAP ™ artery XR S0122 Osteoclastoma-normalized A Osteoclastoma bone disease pBLUESCRIPT ™ S0124 Smooth muscle-edited A Smooth muscle Pulmanary Cell Line UNI-ZAP ™ artery XR S0126 Osteoblasts Osteoblasts Knee Cell Line UNI-ZAP ™ XR S0132 Epithelial-TNFa and INF induced Airway Epithelial UNI-ZAP ™ XR S0134 Apoptotic T-cell apoptotic cells Cell Line UNI-ZAP ™ XR S0136 PERM TF274 stromal cell Bone marrow Cell Line LAMBDA ZAP ™ II S0140 eosinophil-IL5 induced eosinophil lung Cell Line UNI-ZAP ™ XR S0142 Macrophage-oxLDL macrophage-oxidized LDL blood Cell Line UNI-ZAP ™ treated XR S0144 Macrophage (GM-CSF treated) Macrophage (GM-CSF UNI-ZAP ™ treated) XR S0146 prostate-edited prostate BPH Prostate UNI-ZAP ™ XR S0148 Normal Prostate Prostate prostate UNI-ZAP ™ XR S0150 LNCAP prostate cell line LNCAP Cell Line Prostate Cell Line UNI-ZAP ™ XR S0152 PC3 Prostate cell line PC3 prostate cell line UNI-ZAP ™ XR S0168 Prostate/LNCAP, subtraction I PC3 prostate cell line pBLUESCRIPT ™ S0174 Prostate-BPH subtracted II Human Prostate BPH pBLUESCRIPT ™ S0176 Prostate, normal, subtraction I Prostate prostate UNI-ZAP ™ XR S0180 Bone Marrow Stroma, TNF&LPS Bone Marrow Stroma, TNF disease UNI-ZAP ™ ind & LPS induced XR S0182 Human B Cell 8866 Human B-Cell 8866 UNI-ZAP ™ XR S0184 7TM Receptor enriched, lib II PBLS, 7TM receptor Other enriched S0188 Prostate, BPH, Lib 2 Human Prostate BPH disease pSport1 S0190 Prostate BPH, Lib 2, subtracted Human Prostate BPH pSport1 S0192 Synovial Fibroblasts (control) Synovial Fibroblasts pSport1 S0194 Synovial hypoxia Synovial Fibroblasts pSport1 S0196 Synovial IL-1/TNF stimulated Synovial Fibroblasts pSport1 S0198 7TM-pbfd PBLS, 7TM receptor pCR II enriched [Invitrogen] S0202 7TM-pbdd PBLS, 7TM receptor pCR II enriched [Invitrogen] S0206 Smooth Muscle-HASTE Smooth muscle Pulmanary Cell Line pBLUESCRIPT ™ normalized artery S0208 Messangial cell, frac 1 Messangial cell pSport1 S0210 Messangial cell, frac 2 Messangial cell pSport1 S0212 Bone Marrow Stromal Cell, Bone Marrow Stromal pSport1 untreated Cell, untreated S0214 Human Osteoclastoma, re- Osteoclastoma bone disease UNI-ZAP ™ excision XR S0216 Neutrophils IL-1 and LPS human neutrophil induced blood Cell Line UNI-ZAP ™ induced XR S0218 Apoptotic T-cell, re-excision apoptotic cells Cell Line UNI-ZAP ™ XR S0220 H. hypothalamus, frac A; re- Hypothalamus Brain ZAP excision EXPRESS ™ S0222 H. Frontal cortex, epileptic; re- H. Brain, Frontal Cortex, Brain disease UNI-ZAP ™ excision Epileptic XR S0228 PSMIX PBLS, 7TM receptor pCR II enriched [Invitrogen] S0242 Synovial Fibroblasts (Il1/TNF), Synovial Fibroblasts pSport1 subt S0250 Human Osteoblasts II Human Osteoblasts Femur disease pCMVSport 2.0 S0252 7TM-PIMIX PBLS, 7TM receptor pCR II enriched [Invitrogen] S0256 7TM-PHMIX PBLS, 7TM receptor pCR II enriched [Invitrogen] S0260 Spinal Cord, re-excision Spinal cord spinal cord UNI-ZAP ™ XR S0262 PYCS Human Antrum (PY_CS) pCR II [Invitrogen] S0264 PPMIX PPMIX (Human Pituitary) Pituitary pCR II [Invitrogen] S0266 PLMIX PLMIX (Human Lung) Lung pCR II [Invitrogen] S0268 PRMIX PRMIX (Human Prostate) prostate pCR II [Invitrogen] S0270 PTMIX PTMIX (Human Thymus) Thymus pCR II [Invitrogen] S0274 PCMIX PCMIX (Human Brain pCR II Cerebellum) [Invitrogen] S0276 Synovial hypoxia-RSF subtracted Synovial fobroblasts Synovial pSport1 (rheumatoid) tissue S0278 H Macrophage (GM-CSF Macrophage (GM-CSF UNI-ZAP ™ treated), re-excision treated) XR S0280 Human Adipose Tissue, re- Human Adipose Tissue UNI-ZAP ™ excision XR S0282 Brain Frontal Cortex, re-excision Brain frontal cortex Brain LAMBDA ZAP ™ II S0284 7TMCTT (Testis) 7TMCTP (Placenta) Testis pCR II [Invitrogen] S0290 H7TMCTB (Brain) 7TMCTB (Brain) Kidney pCR II [Invitrogen] S0292 Osteoarthritis (OA-4) Human Osteoarthritic Bone disease pSport1 Cartilage S0294 Larynx tumor Larynx tumor Larynx, vocal disease pSport1 cord S0296 Normal lung Normal lung Lung pSport1 S0298 Bone marrow stroma, treated Bone marrow Bone marrow pSport1 stroma, treatedSB S0300 Frontal lobe, dementia; re-excision Frontal Lobe Brain UNI-ZAP ™ dementia/Alzheimer''s XR S0306 Larynx normal #10 261-273 Larynx normal pSport1 S0308 Spleen/normal Spleen normal pSport1 S0310 Normal trachea Normal trachea pSport1 S0312 Human osteoarthritic; fraction II Human osteoarthritic disease pSport1 cartilage S0314 Human osteoarthritis; fraction I Human osteoarthritic disease pSport1 cartilage S0316 Human Normal Human Normal Cartilage pSport1 Cartilage, Fraction I S0318 Human Normal Cartilage Human Normal Cartilage pSport1 Fraction II S0320 Human Larynx Larynx Epiglottis pSport1 S0322 Siebben Polyposis Siebben Polyposis pSport1 S0324 Human Brain Brain Cerebellum pSport1 S0326 Mammary Gland Mammary Gland Whole pSport1 mammary gland S0328 Palate carcinoma Palate carcinoma Uvula disease pSport1 S0330 Palate normal Palate normal Uvula pSport1 S0332 Pharynx carcinoma Pharynx carcinoma Hypopharynx pSport1 S0334 Human Normal Cartilage Human Normal Cartilage pSport1 Fraction III S0336 Human Normal Cartilage Human Normal Cartilage pSport1 Fraction IV S0338 Human Osteoarthritic Cartilage Human osteoarthritic disease pSport1 Fraction III cartilage S0340 Human Osteoarthritic Cartilage Human osteoarthritic disease pSport1 Fraction IV cartilage S0342 Adipocytes; re-excision Human Adipocytes from UNI-ZAP ™ Osteoclastoma XR S0344 Macrophage-oxLDL; re-excision macrophage-oxidized LDL blood Cell Line UNI-ZAP ™ treated XR S0346 Human Amygdala; re-excision Amygdala UNI-ZAP ™ XR S0348 Cheek Carcinoma Cheek Carcinoma disease pSport1 S0350 Pharynx Carcinoma Pharynx carcinoma Hypopharynx disease pSport1 S0352 Larynx Carcinoma Larynx carcinoma disease pSport1 S0354 Colon Normal II Colon Normal Colon pSport1 S0356 Colon Carcinoma Colon Carcinoma Colon disease pSport1 S0358 Colon Normal III Colon Normal Colon pSport1 S0360 Colon Tumor II Colon Tumor Colon disease pSport1 S0362 Human Gastrocnemius Gastrocnemius muscle pSport1 S0364 Human Quadriceps Quadriceps muscle pSport1 S0366 Human Soleus Soleus Muscle pSport1 S0368 Human Pancreatic Langerhans Islets of Langerhans pSport1 S0370 Larynx carcinoma II Larynx carcinoma disease pSport1 S0372 Larynx carcinoma III Larynx carcinoma disease pSport1 S0374 Normal colon Normal colon pSport1 S0376 Colon Tumor Colon Tumor disease pSport1 S0378 Pancreas normal PCA4 No Pancreas Normal PCA4 No pSport1 S0380 Pancreas Tumor PCA4 Tu Pancreas Tumor PCA4 Tu disease pSport1 S0382 Larynx carcinoma IV Larynx carcinoma disease pSport1 S0384 Tongue carcinoma Tongue carcinoma disease pSport1 S0386 Human Whole Brain, re-excision Whole brain Brain ZAP EXPRESS ™ S0388 Human Human Hypothalamus, disease UNI-ZAP ™ Hypothalamus, schizophrenia, re- Schizophrenia XR excision S0390 Smooth muscle, control; re- Smooth muscle Pulmanary Cell Line UNI-ZAP ™ excision artery XR S0392 Salivary Gland Salivary gland; normal pSport1 S0394 Stomach; normal Stomach; normal pSport1 S0396 Uterus; normal Uterus; normal pSport1 S0398 Testis; normal Testis; normal pSport1 S0400 Brain; normal Brain; normal pSport1 S0402 Adrenal Gland, normal Adrenal gland; normal pSport1 S0404 Rectum normal Rectum, normal pSport1 S0406 Rectum tumour Rectum tumour pSport1 S0408 Colon, normal Colon, normal pSport1 S0410 Colon, tumour Colon, tumour pSport1 S0412 Temporal cortex-Alzheizmer; Temporal cortex, alzheimer disease Other subtracted S0414 Hippocampus, Alzheimer Hippocampus, Alzheimer Other Subtracted Subtracted S0418 CHME Cell Line; treated 5 hrs CHME Cell Line; treated pCMVSport 3.0 S0420 CHME Cell Line, untreated CHME Cell line, untreatetd pSport1 S0422 Mo7e Cell Line GM-CSF treated Mo7e Cell Line GM-CSF pCMVSport 3.0 (1 ng/ml) treated (1 ng/ml) S0424 TF-1 Cell Line GM-CSF Treated TF-1 Cell Line GM-CSF pSport1 Treated S0426 Monocyte activated; re-excision Monocyte-activated blood Cell Line UNI-ZAP ™ XR S0428 Neutrophils control; re-excision human neutrophils blood Cell Line UNI-ZAP ™ XR S0430 Aryepiglottis Normal Aryepiglottis Normal pSport1 S0432 Sinus piniformis Tumour Sinus piniformis Tumour pSport1 S0434 Stomach Normal Stomach Normal disease pSport1 S0436 Stomach Tumour Stomach Tumour disease pSport1 S0438 Liver Normal Met5No Liver Normal Met5No pSport1 S0440 Liver Tumour Met 5 Tu Liver Tumour pSport1 S0442 Colon Normal Colon Normal pSport1 S0444 Colon Tumor Colon Tumour disease pSport1 S0446 Tongue Tumour Tongue Tumour pSport1 S0448 Larynx Normal Larynx Normal pSport1 S0450 Larynx Tumour Larynx Tumour pSport1 S0452 Thymus Thymus pSport1 S0454 Placenta Placenta Placenta pSport1 S0456 Tongue Normal Tongue Normal pSport1 S0458 Thyroid Normal (SDCA2 No) Thyroid normal pSport1 S0460 Thyroid Tumour Thyroid Tumour pSport1 S0462 Thyroid Thyroiditis Thyroid Thyroiditis pSport1 S0464 Larynx Normal Larynx Normal pSport1 S0466 Larynx Tumor Larynx Tumor disease pSport1 S0468 Ea.hy.926 cell line Ea.hy.926 cell line pSport1 S0470 Adenocarcinoma PYFD disease pSport1 S0472 Lung Mesothelium PYBT pSport1 S0474 Human blood platelets Platelets Blood Other platelets S0665 Human Amygdala; re-excission Amygdala UNI-ZAP ™ XR S3012 Smooth Muscle Serum Treated, Smooth muscle Pulmanary Cell Line pBLUESCRIPT ™ Norm artery S3014 Smooth muscle, serum Smooth muscle Pulmanary Cell Line pBLUESCRIPT ™ induced, re-exc artery S3018 TH1 cells TH1 cells UNI-ZAP ™ XR S6014 H. hypothalamus, frac A Hypothalamus Brain ZAP EXPRESS ™ S6016 H. Frontal Cortex, Epileptic H. Brain, Frontal Cortex, Brain disease UNI-ZAP ™ Epileptic XR S6022 H. Adipose Tissue Human Adipose Tissue UNI-ZAP ™ XR S6024 Alzheimers, spongy change Alzheimer''s/Spongy Brain disease UNI-ZAP ™ change XR S6026 Frontal Lobe, Dementia Frontal Lobe Brain UNI-ZAP ™ dementia/Alzheimer''s XR S6028 Human Manic Depression Tissue Human Manic depression Brain disease UNI-ZAP ™ tissue XR T0001 Human Brown Fat Brown Fat pBLUESCRIPT ™ SK− T0002 Activated T-cells Activated T-Cell, PBL Blood Cell Line pBLUESCRIPT ™ fraction SK− T0003 Human Fetal Lung Human Fetal Lung pBLUESCRIPT ™ SK− T0004 Human White Fat Human White Fat pBLUESCRIPT ™ SK− T0006 Human Pineal Gland Human Pinneal Gland pBLUESCRIPT ™ SK− T0007 Colon Epithelium Colon Epithelium pBLUESCRIPT ™ SK− T0008 Colorectal Tumor Colorectal Tumor disease pBLUESCRIPT ™ SK− T0010 Human Infant Brain Human Infant Brain Other T0023 Human Pancreatic Carcinoma Human Pancreatic disease pBLUESCRIPT ™ Carcinoma SK− T0039 HSA 172 Cells Human HSA172 cell line pBLUESCRIPT ™ SK− T0040 HSC172 cells SA172 Cells pBLUESCRIPT ™ SK− T0041 Jurkat T-cell G1 phase Jurkat T-cell pBLUESCRIPT ™ SK− T0042 Jurkat T-Cell, S phase Jurkat T-Cell Line pBLUESCRIPT ™ SK− T0047 T lymphocytes >70 T lymphocytes >70 pBLUESCRIPT ™ SK− T0048 Human Aortic Endothelium Human Aortic Endothilium pBLUESCRIPT ™ SK− T0049 Aorta endothelial cells + TNF-a Aorta endothelial cells pBLUESCRIPT ™ SK− T0060 Human White Adipose Human White Fat pBLUESCRIPT ™ SK− T0067 Human Thyroid Human Thyroid pBLUESCRIPT ™ SK− T0067 Human Thyroid Human Thyroid PPBLUESCRIPT ™ SK− T0068 Normal Ovary, Premenopausal Normal Ovary, pBLUESCRIPT ™ Premenopausal SK− T0069 Human Uterus, normal Human Uterus, normal pBLUESCRIPT ™ SK− T0070 Human Adrenal Gland Human Adrenal Gland pBLUESCRIPT ™ SK− T0071 Human Bone Marrow Human Bone Marrow pBLUESCRIPT ™ SK− T0074 Human Adult Retina Human Adult Retina pBluescriptISK− T0078 Human Liver, normal adult Human Liver, normal Adult pBLUESCRIPT ™ SK− T0079 Human Kidney, normal Adult Human Kidney, normal pBLUESCRIPT ™ Adult SK− T0082 Human Adult Retina Human Adult Retina pBLUESCRIPT ™ SK− T0082 Human Adult Retina Human Adult Retina PPBLUESCRIPT ™ SK− T0086 Human Pancreatic Carcinoma -- Human Pancreatic disease pBLUESCRIPT ™ Screened Carcinoma SK− T0087 Alzheimer''s, exon trap, 712P disease pAMP T0090 Liver, normal pBLUESCRIPT ™ SK− T0091 Liver, hepatocellular carcinoma pBLUESCRIPT ™ SK− T0103 Human colon carcinoma (HCC) pBLUESCRIPT ™ cell line SK− T0104 HCC cell line metastisis to liver pBLUESCRIPT ™ SK− T0109 Human (HCC) cell line liver pBLUESCRIPT ™ (mouse) metastasis, remake SK− T0110 Human colon carcinoma (HCC) pBLUESCRIPT ™ cell line, remake SK− T0112 Human (Caco-2) cell line, pBLUESCRIPT ™ adenocarcinoma, colon SK− T0114 Human (Caco-2) cell line, pBLUESCRIPT ™ adenocarcinoma, colon, remake SK− T0115 Human Colon Carcinoma (HCC) pBLUESCRIPT ™ cell line SK−

Table 5

Table 5 provides a key to the OMIM reference identification numbers disclosed in Table 1B.1, column 9. OMIM reference identification numbers (Column 1) were derived from Online Mendelian Inheritance in Man (Online Mendelian Inheritance in Man, OMIM. McKusick-Nathans Institute for Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine, (Bethesda, Md.) 2000 (world wide web at ncbi.nlm.nih.gov/omim/). Column 2 provides diseases associated with the cytologic band disclosed in Table 1B.1, column 8, as determined using the Morbid Map database.

TABLE 5 OMIM Reference Description 100678 ACAT2 deficiency 100690 Myasthenic syndrome, slow-channel congenital, 601462 100710 Myasthenic syndrome, slow-channel congenital, 601462 100730 Myasthenia gravis, neonatal transient 101000 Meningioma, NF2-related, sporadic Schwannoma, sporadic 102200 Somatotrophinoma 102480 Male infertility due to acrosin deficiency 102540 Cardiomyopathy, idiopathic dilated 102578 Leukemia, acute promyelocytic, PML/RARA type 102700 Severe combined immunodeficiency due to ADA deficiency 102770 Myoadenylate deaminase deficiency 102772 [AMP deaminase deficiency, erythrocytic] 103000 Hemolytic anemia due to adenylate kinase deficiency 103050 Autism, succinylpurinemic 103581 Albright hereditary osteodystrophy-2 103600 [Dysalbuminemic hyperthyroxinemia] 103720 Alcoholism, susceptibility to 103850 Aldolase A deficiency 103950 Emphysema due to alpha-2-macroglobulin deficiency 104150 [AFP deficiency, congenital] 104170 NAGA deficiency, mild 104311 Alzheimer disease-3 104500 Amelogenesis imperfecta-2, hypoplastic local type 104770 Amyloidosis, secondary, susceptibility to 105580 Anal canal carcinoma 105600 Dyserythropoietic anemia, congenital, type III 106100 Angioedema, hereditary 106150 Hypertension, essential, susceptibility to 106165 Hypertension, essential, 145500 106180 Myocardial infarction, susceptibility to 106210 Peters anomaly 106300 Ankylosing spondylitis 107250 Anterior segment mesenchymal dysgenesis 107271 CD59 deficiency 107300 Antithrombin III deficiency 107470 Atypical mycobacterial infection, familial disseminated, 209950 107670 Apolipoprotein A-II deficiency 107680 ApoA-I and apoC-III deficiency, combined 107720 Hypertriglyceridemia 107741 Hyperlipoproteinemia, type III 107776 Colton blood group, 110450 107777 Diabetes insipidus, nephrogenic, autosomal recessive, 222000 107910 Virilization, maternal and fetal, from placental aromatase deficiency 107970 Arrhythmogenic right ventricular dysplasia-1 108120 Distal arthrogryposis-1 108725 Atherosclerosis, susceptibility to 108730 Brody myopathy, 601003 108800 Atrial septal defect, secundum type 108962 Hypertension, salt-resistant 108985 Atrophia areata 109150 Machado-Joseph disease 109270 Renal tubular acidosis, distal, 179800 109400 Basal cell nevus syndrome 109560 Leukemia/lymphoma, B-cell, 3 109565 Lymphoma, B-cell 109690 Asthma, nocturnal, susceptibility to 109700 Hemodialysis-related amyloidosis 110100 Blepharophimosis, epicanthus inversus, and ptosis, type 1 110700 Vivax malaria, susceptibility to 112250 Bone dysplasia with medullary fibrosarcoma 112261 Fibrodysplasia ossificans progressiva 112262 Fibrodysplasia ossificans progressiva, 135100 112410 Hypertension with brachydactyly 113100 Brachydactyly, type C 113520 Hyperleucinemia-isoleucinemia or hypervalinemia 113721 Breast cancer 113811 Epidermolysis bullosa, generalized atrophic benign, 226650 113900 Heart block, progressive familial, type I 114130 Osteoporosis 114208 Malignant hyperthermia susceptibility 5, 601887 114240 Muscular dystrophy, limb-girdle, type 2A, 253600 114290 Campomelic dysplasia with autosomal sex reversal 114350 Leukemia, acute myeloid 114400 Lynch cancer family syndrome II 114550 Hepatocellular carcinoma 114835 Monocyte carboxyesterase deficiency 115200 Cardiomyopathy, dilated, 1A 115470 Cat eye syndrome 115500 Acatalasemia 115650 Cataract, anterior polar-1 115660 Cataract, cerulean, type 1 115665 Cataract, congenital, Volkmann type 116600 Cataract, posterior polar 116800 Cataract, Marner type 116806 Colorectal cancer 116860 Cavernous angiomatous malformations 117700 [Hypoceruloplasminemia, hereditary] 118210 Charcot-Marie-Tooth neuropathy-2A 118425 Myotonia congenita, dominant, 160800 118470 [CETP deficiency] 118485 Polycystic ovary syndrome with hyperandrogenemia 118504 Epilepsy, benign neonatal, type 1, 121200 118511 Schizophrenia, neurophysiologic defect in 118800 Choreoathetosis, familial paroxysmal 119300 van der Woude syndrome 119530 Orofacial cleft-1 120070 Alport syndrome, autosomal recessive, 203780 120110 Metaphyseal chondrodysplasia, Schmid type 120120 Epidermolysis bullosa dystrophica, dominant, 131750 120131 Alport syndrome, autosomal recessive, 203780 120140 Osteoarthrosis, precocious 120150 Osteogenesis imperfecta, 4 clinical forms, 166200, 166210, 259420, 166220 120160 Osteogenesis imperfecta, 4 clinical forms, 166200, 166210, 259420, 166220 120180 Ehlers-Danlos syndrome, type III 120190 Ehlers-Danlos syndrome, type I, 130000 120215 Ehlers-Danlos syndrome, type I, 130000 120220 Bethlem myopathy, 158810 120240 Bethlem myopathy, 158810 120260 Epiphyseal dysplasia, multiple, type 2, 600204 120280 Stickler syndrome, type III 120290 OSMED syndrome, 215150 120435 Muir-Torre syndrome, 158320 120436 Muir-Torre family cancer syndrome, 158320 120550 C1q deficiency, type A 120570 C1q deficiency, type B 120575 C1q deficiency, type C 120580 C1r/C1s deficiency, combined 120620 SLE susceptibility 120700 C3 deficiency 120810 C4 deficiency 120820 C4 deficiency 120900 C5 deficiency 120920 Measles, susceptibility to 120940 C9 deficiency 120950 C8 deficiency, type I 120960 C8 deficiency, type II 121011 Deafness, autosomal dominant 3, 601544 121014 Heterotaxia, visceroatrial, autosomal recessive 121050 Contractural arachnodactyly, congenital 121300 Coproporphyria 121360 Myeloid leukemia, acute, M4Eo subtype 121800 Corneal dystrophy, crystalline, Schnyder 122720 Nicotine addiction, protection from 123000 Craniometaphyseal dysplasia 123100 Craniosynostosis, type 1 123101 Craniosynostosis, type 2 123270 [Creatine kinase, brain type, ectopic expression of] 123580 Cataract, congenital, autosomal dominant 123620 Cataract, cerulean, type 2, 601547 123660 Cataract, Coppock-like 123829 Melanoma 123940 White sponge nevus, 193900 124020 Mephenytoin poor metabolizer 124030 Parkinsonism, susceptibility to 124200 Darier disease (keratosis follicularis) 125264 Leukemia, acute nonlymphocytic 125270 Porphyria, acute hepatic 125370 Dentatorubro-pallidoluysian atrophy 125490 Dentinogenesis imperfecta-1 125660 Myopathy, desminopathic 125852 Insulin-dependent diabetes mellitus-2 126060 Anemia, megaloblastic, due to DHFR deficiency 126090 Hyperphenylalaninemia due to pterin-4a-carbinolamine dehydratase deficiency, 264070 126337 Myxoid liposarcoma 126340 Xeroderma pigmentosum, group D, 278730 126391 DNA ligase I deficiency 126451 Schizophrenia, susceptibility to 126452 Autonomic nervous system dysfunction 126600 Doyne honeycomb retinal dystrophy 126650 Chloride diarrhea, congenital, Finnish type, 214700 128100 Dystonia-1, torsion 129010 Neuropathy, congenital hypomyelinating, 1 129490 Ectodermal dysplasia-3, anhidrotic 129500 Ectodermal dysplasia, hidrotic 129900 EEC syndrome-1 130410 Glutaricaciduria, type IIB 130500 Elliptocytosis-1 130650 Beckwith-Wiedemann syndrome 131100 Multiple endocrine neoplasia I 131195 Hereditary hemorrhagic telangiectasia-1, 187300 131210 Atherosclerosis, susceptibility to 131242 Shah-Waardenburg syndrome, 277580 131244 Hirschsprung disease-2, 600155 131400 Eosinophilia, familial 131440 Eosinophilic myeloproliferative disorder 131950 Epidermolysis bullosa, Ogna type 132700 Cylindromatosis 132800 Basal cell carcinoma 132810 Diphenylhydantoin toxicity 133170 Erythremia 133171 [Erythrocytosis, familial], 133100 133200 Erythrokeratodermia variabilis 133430 Breast cancer 133450 Neuroepithelioma 133510 Trichothiodystrophy 133530 Xeroderma pigmentosum, group G, 278780 133550 Dicarboxylicaminoaciduria, 222730 133700 Chondrosarcoma, 215300 133701 Exostoses, multiple, type 2 133780 Vitreoretinopathy, exudative, familial 134370 Membroproliferative glomerulonephritis 134570 Factor XIIIA deficiency 134580 Factor XIIIB deficiency 134637 Autoimmune lymphoproliferative syndrome 134638 Systemic lupus erythematosus, susceptibility, 152700 134790 Hyperferritinemia-cataract syndrome, 600886 134797 Shprintzen-Goldberg syndrome, 182212 134820 Dysfibrinogenemia, alpha type, causing bleeding diathesis 134830 Dysfibrinogenemia, beta type 134850 Dysfibrinogenemia, gamma type 134934 Thanatophoric dysplasia, types I and II, 187600 135300 Fibromatosis, gingival 135600 Ehlers-Danlos syndrome, type X 135700 Fibrosis of extraocular muscles, congenital, 1 135940 Ichthyosis vulgaris, 146700 136132 [Fish-odor syndrome], 602079 136350 Pfeiffer syndrome, 101600 136435 Ovarian dysgenesis, hypergonadotropic, with normal karyotype, 233300 136530 Male infertility, familial 136550 Macular dystrophy, North Carolina type 136836 Fucosyltransferase-6 deficiency 137350 Amyloidosis, Finnish type, 105120 137600 Iridogoniodysgenesis syndrome 138030 [Hyperproglucagonemia] 138033 Diabetes mellitus, type II 138040 Cortisol resistance 138079 Hyperinsulinism, familial, 602485 138130 Hyperinsulinism-hyperammonemia syndrome 138140 Glucose transport defect, blood-brain barrier 138160 Diabetes mellitus, noninsulin-dependent 138190 Diabetes mellitus, noninsulin-dependent 138250 P5CS deficiency 138300 Hemolytic anemia due to glutathione reductase deficiency 138320 Hemolytic anemia due to glutathione peroxidase deficiency 138491 Startle disease, autosomal recessive 138570 Non-insulin dependent diabetes mellitus, susceptibility to 138571 Glycogen synthase, liver, deficiency of, 240600 138700 [Apolipoprotein H deficiency] 138720 Bernard-Soulier syndrome, type B 138760 [Glyoxalase II deficiency] 138971 Kostmann neutropenia, 202700 138981 Pulmonary alveolar proteinosis, 265120 139130 Hypertension, essential, susceptibility to, 145500 139150 Basal cell carcinoma 139190 Gigantism due to GHRF hypersecretion 139191 Growth hormone deficient dwarfism 139250 Isolated growth hormone deficiency, Illig type with absent GH and Kowarski type with bioinactive GH 139320 Pituitary ACTH secreting adenoma 139330 Night blindness, congenital stationary 139350 Epidermolytic hyperkeratosis, 113800 139360 Pituitary ACTH-secreting adenoma 140100 [Anhaptoglobinemia] 141750 Alpha-thalassemia/mental retardation syndrome, type 1 141800 Methemoglobinemias, alpha- 141850 Thalassemia, alpha- 141900 Methemoglobinemias, beta- 142000 Thalassemia due to Hb Lepore 142200 HPFH, nondeletion type A 142250 HPFH, nondeletion type G 142270 Hereditary persistence of fetal hemoglobin 142335 Hereditary persistence of fetal hemoglobin, heterocellular, Indian type 142360 Thrombophilia due to heparin cofactor II deficiency 142380 Hepatocellular carcinoma 142470 [Hereditary persistence of fetal hemoglobin, heterocellular] 142600 Hemolytic anemia due to hexokinase deficiency 142640 Thrombophilia due to elevated HRG 142680 Periodic fever, familial 142857 Pemphigoid, susceptibility to 142858 Beryllium disease, chronic, susceptibility to 142946 Holoprosencephaly-4 142959 Hand-foot-uterus syndrome, 140000 142989 Synpolydactyly, type II, 186000 143100 Huntington disease 143200 Wagner syndrome 143890 Hypercholesterolemia, familial 144120 Hyperimmunoglobulin G1 syndrome 144200 Epidermolytic palmoplantar keratoderma 145001 Hyperparathyroidism-jaw tumor syndrome 145260 Pseudohypoaldosteronism, type II 145410 Opitz G syndrome, type II 145505 Hypertension, essential 145981 Hypocalciuric hypercalcemia, type II 146150 Hypomelanosis of Ito 146200 Hypoparathyroidism, familial 146740 Neutropenia, alloimmune neonatal 146760 [IgG receptor I, phagocytic, familial deficiency of] 146790 Lupus nephritis, susceptibility to 147020 Agammaglobulinemia, 601495 147050 Atopy 147061 Allergy and asthma susceptibility 147110 IgG2 deficiency, selective 147141 Leukemia, acute lymphoblastic 147200 [Kappa light chain deficiency] 147280 Hepatocellular carcinoma 147440 Growth retardation with deafness and mental retardation 147450 Amytrophic lateral sclerosis, due to SOD1 deficiency, 105400 147545 Diabetes mellitus, noninsulin-dependent 147570 Interferon, immune, deficiency 147575 Myelodysplastic syndrome, preleukemic 147660 Interferon, alpha, deficiency 147670 Rabson-Mendenhall syndrome 147680 Severe combined immunodeficiency due to IL2 deficiency 147730 Interleukin-2 receptor, alpha chain, deficiency of 147781 Atopy, susceptibility to 147790 Leukemia, acute lymphocytic, with 4/11 translocation 147791 Jacobsen syndrome 148040 Epidermolysis bullosa simplex, Koebner, Dowling-Meara, and Weber-Cockayne types, 131900, 131760, 131800 148041 Pachyonychia congenita, Jadassohn-Lewandowsky type, 167200 148043 Meesmann corneal dystrophy, 122100 148065 White sponge nevus, 193900 148066 Epidermolysis bullosa simplex, Koebner, Dowling-Meara, and Weber-Cockayne types, 131900, 131760, 131800 148067 Nonepidermolytic palmoplantar keratoderma, 600962 148069 Pachyonychia congenita, Jackson-Lawler type, 167210 148070 Liver disease, susceptibility to, from hepatotoxins or viruses 148080 Epidermolytic hyperkeratosis, 113800 148370 Keratolytic winter erythema 148500 Tylosis with esophageal cancer 148900 Klippel-Feil syndrome with laryngeal malformation 150000 Exertional myoglobinuria due to deficiency of LDH-A 150100 Lactate dehydrogenase-B deficiency 150200 [Placental lactogen deficiency] 150210 Lactoferrin-deficient neutrophils, 245480 150230 Langer-Giedion syndrome 150240 Cutis laxa, marfanoid neonatal type 150250 Larsen syndrome, autosomal dominant 150270 Laryngeal adductor paralysis 150292 Epidermolysis bullosa, Herlitz junctional type, 226700 150310 Epidermolysis bullosa, Herlitz junctional type, 226700 151385 Leukemia, acute myeloid 151390 Leukemia, acute T-cell 151400 Leukemia/lymphoma, B-cell, 1 151410 Leukemia, chronic myeloid 151440 Leukemia, T-cell acute lymphoblastoid 151670 Hepatic lipase deficiency 152200 Coronary artery disease, susceptibility to 152427 Long QT syndrome-2 152445 Vohwinkel syndrome, 124500 152760 Hypogonadotropic hypogonadism due to GNRH deficiency, 227200 152780 Hypogonadism, hypergonadotropic 152790 Precocious puberty, male, 176410 153454 Ehlers-Danlos syndrome, type VI, 225400 153455 Cutis laxa, recessive, type I, 219100 153700 Macular dystrophy, vitelliform type 153840 Macular dystrophy, atypical vitelliform 153880 Macular dystrophy, dominant cystoid 153900 Stargardt disease-2 154275 Malignant hyperthermia susceptibility 2 154276 Malignant hyperthermia susceptibility 3 154400 Acrofacial dysostosis, Nager type 154500 Treacher Collins mandibulofacial dysostosis 154545 Chronic infections, due to opsonin defect 154550 Carbohydrate-deficient glycoprotein syndrome, type Ib, 602579 154705 Marfan syndrome, type II 155555 [Red hair/fair skin] 155600 Malignant melanoma, cutaneous 156225 Muscular dystrophy, congenital merosin-deficient 156232 Mesomelic dysplasia, Kantaputra type 156570 Methylcobalamin deficiency, cbl G type 156600 Microcoria, congenital 156845 Tietz syndrome, 103500 156850 Cataract, congenital, with microphthalmia 157140 Dementia, frontotemporal, with parkinsonism, 601630 157147 Abetalipoproteinemia, 200100 157170 Holoprosencephaly-2 157640 PEO with mitochondrial DNA deletions, type 1 157655 Lactic acidosis due to defect in iron-sulfur cluster of complex I 157900 Moebius syndrome 158590 Spinal muscular atrophy-4 159000 Muscular dystrophy, limb-girdle, type 1A 159001 Muscular dystrophy, limb-girdle, type 1B 159440 Charcot-Marie-Tooth neuropathy-1B, 118200 159555 Leukemia, myeloid/lymphoid or mixed-lineage 159595 Leukemia, transient, of Down syndrome 160760 Cardiomyopathy, familial hypertrophic, 1, 192600 160777 Griscelli disease, 214450 160781 Cardiomyopathy, hypertrophic, mid-left ventricular chamber type 160900 Myotonic dystrophy 160980 Carney myxoma-endocrine complex 161015 Mitochondrial complex I deficiency, 252010 162100 Neuralgic amyotrophy with predilection for brachial plexus 162150 Obestiy with impaired prohormone processing, 600955 162200 Neurofibromatosis, type 1 162400 Neuropathy, hereditary sensory and autonomic, type 1 163729 Hypertension, pregnancy-induced 163890 Parkinson disease, type 1, 601508 163950 Noonan syndrome-1 164009 Leukemia, acute promyelocytic, NUMA/RARA type 164040 Leukemia, acute promyelocytic, NPM/RARA type 164160 Obesity, severe, due to leptin deficiency 164200 Oculodentodigital dysplasia 164500 Spinocerebellar ataxia-7 164731 Ovarian carcinoma, 167000 164759 Ovarian carcinoma 164761 Medullary thyroid carcinoma, 155240 164770 Myeloid malignancy, predisposition to 164790 Colorectal cancer 164860 Renal cell carcinoma, papillary, familial and sporadic 164920 Piebaldism 164953 Liposarcoma 165240 Pallister-Hall syndrome, 146510 165320 Hepatocellular carcinoma 165500 Optic atrophy 1 166600 Osteopetrosis, AD, type II 166800 Otosclerosis 167000 Ovarian cancer, serous 167250 Paget disease of bone 167409 Optic nerve coloboma with renal disease, 120330 167410 Rhabdomyosarcoma, alveolar, 268220 167415 Hypothyroidism, congenital, due to thyroid dysgenesis or hypoplasia 168000 Paraganglioma, familial nonchromaffin, 1 168360 Paraneoplastic sensory neuropathy 168450 Hypoparathyroidism, autosomal dominant 168461 Multiple myeloma, 254250 168468 Metaphyseal chondrodysplasia, Murk Jansen type, 156400 168470 Humoral hypercalcemia of malignancy 168500 Parietal foramina 169600 Hailey-Hailey disease 170261 Bare lymphocyte syndrome, type I, due to TAP2 deficiency 170500 Myotonia congenita, atypical acetazolamide-responsive 170650 Periodontitis, juvenile 170995 Zellweger syndrome-2 171050 Colchicine resistance 171060 Cholestasis, progressive familial intrahepatic, type III, 602347 171190 Hypertension, essential, 145500 171650 Lysosomal acid phosphatase deficiency 171760 Hypophosphatasia, adult, 146300 171860 Hemolytic anemia due to phosphofructokinase deficiency 172400 Hemolytic anemia due to glucosephosphate isomerase deficiency 172411 Colorectal cancer, resistance to 172430 Enolase deficiency 172471 Glycogenosis, hepatic, autosomal 172490 Phosphorylase kinase deficiency of liver and muscle, 261750 173110 Pituitary hormone deficiency, combined 173350 Plasminogen Tochigi disease 173360 Thrombophilia due to excessive plasminogen activator inhibitor 173370 Plasminogen activator deficiency 173470 Glanzmann thrombasthenia, type B 173610 Platelet alpha/delta storage pool deficiency 173850 Polio, susceptibility to 173870 Xeroderma pigmentosum 173910 Polycystic kidney disease, adult, type II 174000 Medullary cystic kidney disease, AD 174810 Osteolysis, familial expansile 174900 Polyposis, juvenile intestinal 175100 Turcot syndrome, 276300 176000 Porphyria, acute intermittent 176100 Porphyria cutanea tarda 176260 Episodic ataxia/myokymia syndrome, 160120 176261 Jervell and Lange-Nielsen syndrome, 220400 176270 Prader-Willi syndrome 176300 [Dystransthyretinemic hyperthyroxinemia] 176310 Leukemia, acute pre-B-cell 176450 Sacral agenesis-1 176640 Creutzfeldt-Jakob disease, 123400 176730 Diabetes mellitus, rare form 176801 Metachromatic leukodystrophy due to deficiency of SAP-1 176830 Obesity, adrenal insufficiency, and red hair 176860 Purpura fulminans, neonatal 176880 Protein S deficiency 176930 Dysprothrombinemia 176943 Apert syndrome, 101200 176947 Selective T-cell defect 176960 Pituitary tumor, invasive 177070 Spherocytosis, hereditary, Japanese type 177400 Apnea, postanesthetic 177900 Psoriasis susceptibility-1 178300 Ptosis, hereditary congenital, 1 178600 Pulmonary hypertension, familial primary 178640 Pulmonary alveolar proteinosis, congenital, 265120 179095 Male infertility 179450 Ragweed sensitivity 179605 Retinitis pigmentosa, digenic 179615 Reticulosis, familial histiocytic, 267700 179616 Severe combined immunodeficiency, B cell-negative, 601457 179755 Renal cell carcinoma, papillary, 1 179820 [Hyperproreninemia] 180020 Retinal cone dystrophy-1 180069 Retinal dystrophy, autosomal recessive, childhood-onset 180071 Retinitis pigmentosa, autosomal recessive 180072 Night blindness, congenital stationary, type 3, 163500 180090 Retinitis pigmentosa, autosomal recessive 180100 Retinitis pigmentosa-1 180104 Retinitis pigmentosa-9 180105 Retinitis pigmentosa-10 180200 Osteosarcoma, 259500 180240 Leukemia, acute promyelocytic 180250 Retinol binding protein, deficiency of 180297 Anemia, hemolytic, Rh-null, suppressor type, 268150 180380 Night blindness, congenital stationery, rhodopsin-related 180381 Oguchi disease-2, 258100 180385 Leukemia, acute T-cell 180721 Retinitis pigmentosa, digenic 180840 Susceptibility to IDDM 180860 Russell-Silver syndrome 180901 Malignant hyperthermia susceptibility 1, 145600 181030 Salivary gland pleomorphic adenoma 181031 Oguchi disease-1, 258100 181405 Scapuloperoneal spinal muscular atrophy, New England type 181430 Scapuloperoneal syndrome, myopathic type 181460 Schistosoma mansoni, susceptibility/resistance to 181510 Schizophrenia 181600 Sclerotylosis 182138 Anxiety-related personality traits 182279 Prader-Willi syndrome 182280 Small-cell cancer of lung 182290 Smith-Magenis syndrome 182380 Glucose/galactose malabsorption 182381 Renal glucosuria, 253100 182452 Lung cancer, small cell 182500 Cataract, congenital 182600 Spastic paraplegia-3A 182601 Spastic paraplegia-4 182860 Pyropoikilocytosis 182870 Spherocytosis-1 182900 Spherocytosis-2 183600 Split hand/foot malformation, type 1 185000 Stomatocytosis I 185430 Atherosclerosis, susceptibility to 185470 Myopathy due to succinate dehydrogenase deficiency 185800 Symphalangism, proximal 186580 Arthrocutaneouveal granulomatosis 186740 Immunodeficiency due to defect in CD3-gamma 186770 Leukemia, T-cell acute lymphocytic 186780 CD3, zeta chain, deficiency 186830 Immunodeficiency, T-cell receptor/CD3 complex 186855 Leukemia-2, T-cell acute lymphoblastic 186860 Leukemia/lymphoma, T-cell 186880 Leukemia/lymphoma, T-cell 186921 Leukemia, T-cell acute lymphoblastic 186940 [CD4(+) lymphocyte deficiency] 187040 Leukemia-1, T-cell acute lymphoblastic 187680 6-mercaptopurine sensitivity 188025 Thrombocytopenia, Paris-Trousseau type 188070 Bleeding disorder due to defective thromboxane A2 receptor 188400 Velocardiofacial syndrome, 192430 188450 Goiter, adolescent multinodular 188540 Hypothyroidism, nongoitrous 188550 Thyroid papillary carcinoma 188826 Sorsby fundus dystrophy, 136900 189800 Preeclampsia/eclampsia 189980 Leukemia, chronic myeloid 190000 Atransferrinemia 190020 Bladder cancer, 109800 190040 Meningioma, SIS-related 190070 Colorectal adenoma 190100 Geniospasm 190160 Thyroid hormone resistance, 274300, 188570 190182 Colon cancer 190195 Ichthyosiform erythroderma, congenital, 242100 190198 Leukemia, T-cell acute lymphoblastic 190300 Tremor, familial essential, 1 190450 Hemolytic anemia due to triosephosphate isomerase deficiency 190605 Triphalangeal thumb-polysyndactyly syndrome 190685 Down syndrome 190900 Colorblindness, tritan 191010 Cardiomyopathy, familial hypertrophic, 3, 115196 191030 Nemaline myopathy-1, 161800 191044 Cardiomyopathy, familial hypertrophic 191045 Cardiomyopathy, familial hypertrophic, 2, 115195 191092 Tuberous sclerosis-2 191100 Tuberous sclerosis-1 191170 Colorectal cancer, 114500 191181 Cervical carcinoma 191290 Segawa syndrome, recessive 191315 Insensitivity to pain, congenital, with anhidrosis, 256800 191540 [Urate oxidase deficiency] 192090 Ovarian carcinoma 192340 Diabetes insipidus, neurohypophyseal, 125700 192500 Jervell and Lange-Nielsen syndrome, 220400 192974 Neonatal alloimmune thrombocytopenia 193100 Hypophosphatemic rickets, autosomal dominant 193235 Vitreoretinopathy, neovascular inflammatory 193300 Renal cell carcinoma 193400 von Willebrand disease 193500 Rhabdomyosarcoma, alveolar, 268220 194070 Wilms tumor, type 1 194071 Wilms tumor, type 2 194190 Wolf-Hirschhorn syndrome 200150 Choreoacanthocytosis 200990 Acrocallosal syndrome 201450 Acyl-CoA dehydrogenase, medium chain, deficiency of 201460 Acyl-CoA dehydrogenase, long chain, deficiency of 201470 Acyl-CoA dehydrogenase, short-chain, deficiency of 201475 VLCAD deficiency 201810 3-beta-hydroxysteroid dehydrogenase, type II, deficiency 201910 Adrenal hyperplasia, congenital, due to 21-hydroxylase deficiency 202110 Adrenal hyperplasia, congenital, due to 17-alpha- hydroxylase deficiency 203100 Waardenburg syndrome/ocular albinism, digenic, 103470 203200 Albinism, ocular, autosomal recessive 203300 Hermansky-Pudlak syndrome 203310 Ocular albinism, autosomal recessive 203500 Alkaptonuria 203740 Alpha-ketoglutarate dehydrogenase deficiency 203750 3-ketothiolase deficiency 203800 Alstrom syndrome 204500 Ceroid-lipofuscinosis, neuronal 2, classic late infantile 205100 Amyotrophic lateral sclerosis, juvenile 205900 Anemia, Diamond-Blackfan 207750 Hyperlipoproteinemia, type Ib 207800 Argininemia 208100 Arthrogryposis multiplex congenita, neurogenic 208250 Jacobs syndrome 208400 Aspartylglucosaminuria 208900 Ataxia-telangiectasia 209900 Bardet-Biedl syndrome 2 209901 Bardet-Biedl syndrome 1 210900 Bloom syndrome 211420 Breast cancer, ductal 212138 Carnitine-acylcarnitine translocase deficiency 214300 Klippel-Feil syndrome 214400 Charcot-Marie-Tooth neuropathy-4A 214500 Chediak-Higashi syndrome 215700 Citrullinemia 216550 Cohen syndrome 216900 Achromatopsia 216950 C1r/C1s deficiency, combined 217000 C2 deficiency 217030 C3b inactivator deficiency 217050 C6 deficiency 217070 C7 deficiency 217095 Conotruncal cardiac anomalies 217300 Cornea plana congenita, recessive 217800 Macular corneal dystrophy 218000 Andermann syndrome 218030 Apparent mineralocorticoid excess, hypertension due to 219800 Cystinosis, nephropathic 221770 Polycystic lipomembranous osteodysplasia with sclerosing leukencephalopathy 221820 Gliosis, familial progressive subcortical 222100 Diabetes mellitus, insulin-dependent-1 222600 Atelosteogenesis II, 256050 222700 Lysinuric protein intolerance 222745 DECR deficiency 222800 Hemolytic anemia due to bisphosphoglycerate mutase deficiency 222900 Sucrose intolerance 223000 Lactase deficiency, adult, 223100 223360 Dopamine-beta-hydroxylase deficiency 223900 Dysautonomia, familial 224100 Congenital dyserythropoietic anemia II 224120 Dyserythropoietic anemia, contenital, type I 225500 Ellis-van Creveld syndrome 226450 Epidermolysis bullosa inversa, junctional 227220 [Eye color, brown] 227400 Thromboembolism susceptibility due to factor V Leiden 227500 Factor VII deficiency 227600 Factor X deficiency 227645 Fanconi anemia, type C 227646 Fanconi anemia, type D 227650 Fanconi anemia, type A 228960 [Kininogen deficiency] 229300 Friedreich ataxia 229600 Fructose intolerance 229700 Fructose-bisphosphatase deficiency 229800 [Fructosuria] 230000 Fucosidosis 230200 Galactokinase deficiency with cataracts 230350 Galactose epimerase deficiency 230400 Galactosemia 230450 Hemolytic anemia due to gamma-glutamylcysteine synthetase deficiency 230800 Gaucher disease 231550 Achalasia-addisonianism-alacrimia syndrome 231670 Glutaricaciduria, type I 231675 Glutaricaciduria, type IIC 231680 Glutaricaciduria, type IIA 231950 Glutathioninuria 232000 Propionicacidemia, type I or pccA type 232050 Propionicacidemia, type II or pccB type 232300 Glycogen storage disease II 232400 Glycogen storage disease IIIa 232500 Glycogen storage disease IV 232600 McArdle disease 232700 Glycogen storage disease VI 232800 Glycogen storage disease VII 233100 [Renal glucosuria] 233700 Chronic granulomatous disease due to deficiency of NCF-1 233710 Chronic granulomatous disease due to deficiency of NCF-2 234000 Factor XII deficiency 234200 Neurodegeneration with brain iron accumulation 235200 Hemochromatosis 235800 [Histidinemia] 236100 Holoprosencephaly-1 236200 Homocystinuria, B6-responsive and nonresponsive types 236250 Homocystinuria due to MTHFR deficiency 236700 McKusick-Kaufman syndrome 236730 Urofacial syndrome 237300 Carbamoylphosphate synthetase I deficiency 238300 Hyperglycinemia, nonketotic, type I 238310 Hyperglycinemia, nonketotic, type II 238600 Chylomicronemia syndrome, familial 238970 HHH syndrome 239100 Van Buchem disease 239500 Hyperprolinemia, type I 240300 Autoimmune polyglandular disease, type I 240400 Scurvy 243500 Isovalericacidemia 245000 Papillon-Lefevre syndrome 245050 Ketoacidosis due to SCOT deficiency 245200 Krabbe disease 245349 Lacticacidemia due to PDX1 deficiency 245900 Norum disease 246450 HMG-CoA lyase deficiency 246530 Leukotriene C4 synthase deficiency 246600 Pancreatic lipase deficiency 246900 Lipoamide dehydrogenase deficiency 247200 Miller-Dieker lissencephaly syndrome 247640 Leukemia, acute lymphoblastic 248510 Mannosidosis, beta- 248600 Maple syrup urine disease, type Ia 248610 Maple syrup urine disease, type II 248611 Maple syrup urine disease, type Ib 249000 Meckel syndrome 249100 Familial Mediterranean fever 249270 Thiamine-responsive megaloblastic anemia 250100 Metachromatic leukodystrophy 250250 Cartilage-hair hypoplasia 250790 Methemoglobinemia due to cytochrome b5 deficiency 250800 Methemoglobinemia, type I 250850 Hypermethioninemia, persistent, autosomal dominant, due to methionine adenosyltransferase I/III deficiency 251000 Methylmalonicaciduria, mutase deficiency type 251170 Mevalonicaciduria 251600 Microphthalmia, autosomal recessive 252500 Mucolipidosis II 252800 Mucopolysaccharidosis Ih 252900 Sanfilippo syndrome, type A 252940 Sanfilippo syndrome, type D 253000 Mucopolysaccharidosis IV A 253200 Maroteaux-Lamy syndrome, several forms 253250 Mulibrey nanism 253270 Multiple carboxylase deficiency, biotin-responsive 253601 Miyoshi myopathy, 254130 253700 Muscular dystrophy, limb-girdle, type 2C 253800 Walker-Warburg syndrome, 236670 254210 Myasthenia gravis, familial infantile 254770 Epilepsy, juvenile myoclonic 254780 Myoclonus epilepsy, Lafora type 255800 Schwartz-Jampel syndrome 256030 Nemaline myopathy-2 256100 Nephronophthisis, juvenile 256540 Galactosialidosis 256550 Sialidosis, type I 256700 Neuroblastoma 256731 Ceroid-lipofuscinosis, neuronal-5, variant late infantile 256850 Giant axonal neuropathy-1 257200 Niemann-Pick disease, type A 257220 Niemann-Pick disease, type C 258501 3-methylglutaconicaciduria, type III 258870 Gyrate atrophy of choroid and retina with ornithinemia, B6 responsive or unresponsive 258900 Oroticaciduria 259700 Osteopetrosis, recessive 259730 Renal tubular acidosis-osteopetrosis syndrome 259770 Osteoporosis-pseudoglioma syndrome 259900 Hyperoxaluria, primary, type 1 261510 Pseudo-Zellweger syndrome 261515 Peroxisomal bifunctional enzyme deficiency 261600 Phenylketonuria 261640 Phenylketonuria due to PTS deficiency 261670 Myopathy due to phosphoglycerate mutase deficiency 262000 Bjornstad syndrome 263200 Polycystic kidney disease, autosomal recessive 263700 Porphyria, congenital erythropoietic 264300 Pseudohermaphroditism, male, with gynecomastia 264470 Adrenoleukodystrophy, pseudoneonatal 264700 Pseudo-vitamin D dependency rickets 1 266100 Pyridoxine dependency with seizures 266150 Pyruvate carboxylase deficiency 266200 Anemia, hemolytic, due to PK deficiency 266300 [Hair color, red] 266600 Inflammatory bowel disease-1 267750 Knobloch syndrome 268800 Sandhoff disease, infantile, juvenile, and adult forms 268900 [Sarcosinemia] 269920 Salla disease 270100 Situs inversus viscerum 270200 Sjogren-Larsson syndrome 270800 Spastic paraplegia-5A 271245 Spinocerebellar ataxia-8, infantile, with sensory neuropathy 271900 Canavan disease 272750 GM2-gangliosidosis, AB variant 272800 Tay-Sachs disease 273300 Male germ cell tumor 273800 Thrombocytopenia, neonatal alloimmune 274180 Thromboxane synthase deficiency 274270 Thymine-uraciluria 274600 Pendred syndrome 275200 Thyroid adenoma, hyperfunctioning 275350 Transcobalamin II deficiency 276000 Pancreatitis, hereditary, 167800 276600 Tyrosinemia, type II 276700 Tyrosinemia, type I 276710 Tyrosinemia, type III 276900 Usher syndrome, type 1A 276901 Usher syndrome, type 2 276902 Usher syndrome, type 3 276903 Usher syndrome, type 1B 276904 Usher syndrome, type 1C 277700 Werner syndrome 277730 Wernicke-Korsakoff syndrome, susceptibility to 277900 Wilson disease 278000 Wolman disease 278250 Wrinkly skin syndrome 278300 Xanthinuria, type I 278700 Xeroderma pigmentosum, group A 278760 Xeroderma pigmentosum, group F 300000 Opitz G syndrome, type I 300008 Nephrolithiasis, type I, 310468 300011 Menkes disease, 309400 300031 Mental retardation, X-linked, FRAXF type 300032 Alpha-thalassemia/mental retardation syndrome, type 2, 301040 300037 Simpson dysmorphia syndrome, 312870 300039 Deafness, X-linked 3, conductive, with stapes fixation, 304400 300044 Wernicke-Korsakoff syndrome, susceptibility to 300046 Mental retardation, X-linked 23, nonspecific 300047 Mental retardation, X-linked 20 300048 Intestinal pseudoobstruction, neuronal, X-linked 300049 Nodular heterotopia, bilateral periventricular 300055 Mental retardation with psychosis, pyramidal signs, and macroorchidism 300062 Mental retardation, X-linked 14 300066 Deafness, X-linked 6, sensorineural 300067 Subcortical laminar heterotopia, X-linked dominant 300071 Night blindness, congenital stationary, type 2 300075 Coffin-Lowry syndrome, 303600 300076 Wood neuroimmunologic syndrome 300077 Mental retardation, X-linked 29 300088 Epilepsy, female restricted, with mental retardation 300100 Adrenoleukodystrophy 300104 Mental retardation, X-linked nonspecific, 309541 300110 Night blindness, congenital stationary, X-linked incomplete, 300071 300121 Subcortical laminal heteropia, X-linked, 300067 300123 Mental retardation with isolated growth hormone deficiency 300126 Dyskeratosis congenita-1, 305000 300127 Mental retardation, X-linked, 60 300136 Diabetes mellitus, insulin-dependent, X-linked, susceptibility to 300300 XLA and isolated growth hormone deficiency, 307200 300310 Agammaglobulinemia, type 2, X-linked 300500 Ocular albinism, Nettleship-Falls type 300600 Ocular albinism, Forsius-Eriksson type 300650 Ocular albinism with sensorineural deafness 301000 Thrombocytopenia, X-linked, 313900 301200 Amelogenesis imperfecta 301201 Amelogenesis imperfecta-3, hypoplastic type 301220 Partington syndrome II 301300 Anemia, sideroblastic/hypochromic 301310 Anemia, sideroblastic, with spinocerebellar ataxia 301500 Fabry disease 301590 Anophthalmos-1 301830 Arthrogryposis, X-linked (spinal muscular atrophy, infantile, X-linked) 301835 Arts syndrome 301845 Bazex syndrome 301900 Borjeson-Forssman-Lehmann syndrome 302060 Noncompaction of left ventricular myocardium, isolated 302350 Nance-Horan syndrome 302801 Charcot-Marie-Tooth neuropathy, X-linked-2, recessive 302950 Chondrodysplasia punctata, X-linked recessive, 302940 302960 Chondrodysplasia punctata, X-linked dominant 303400 Cleft palate, X-linked 303630 Alport syndrome, 301050 303631 Leiomyomatosis, diffuse, with Alport syndrome 303700 Colorblindness, blue monochromatic 303800 Colorblindness, deutan 303900 Colorblindness, protan 304020 Cone dystrophy, progressive X-linked, 1 304040 Charcot-Marie-Tooth neuropathy, X-linked-1, dominant, 302800 304050 Aicardi syndrome 304110 Craniofrontonasal dysplasia 304340 Mental retardation, X-linked, syndromic-5, with Dandy- Walker malformation, basal ganglia disease, and seizures 304500 Deafness, X-linked 2, perceptive congenital 304700 Mohr-Tranebjaerg syndrome 304800 Diabetes insipidus, nephrogenic 305100 Anhidrotic ectodermal dysplasia 305400 Aarskog-Scott syndrome 305435 Heterocellular hereditary persistence of fetal hemoglobin, Swiss type 305450 FG syndrome 305900 Favism 306000 Glycogenosis, X-linked hepatic, type I 306100 Gonadal dysgenesis, XY female type 306400 Chronic granulomatous disease, X-linked 306700 Hemophilia A 306900 Hemophilia B 306995 [Homosexuality, male] 307150 Hypertrichosis, congenital generalized 307700 Hypoparathyroidism, X-linked 307800 Hypophosphatemia, hereditary 308000 HPRT-related gout 308230 Immunodeficiency, X-linked, with hyper-IgM 308240 Lymphoproliferative syndrome, X-linked 308300 Incontinentia pigmenti, sporadic type 308310 Incontinentia pigmenti, familial 308380 Severe combined immunodeficiency, X-linked, 300400 308700 Kallmann syndrome 308800 Keratosis follicularis spinulosa decalvans 308840 Spastic paraplegia, 312900 309000 Lowe syndrome 309200 Manic-depressive illness, X-linked 309300 Megalocornea, X-linked 309470 Mental retardation, X-linked, syndromic-3, with spastic diplegia 309500 Renpenning syndrome-1 309510 Mental retardation, X-linked, syndromic-1, with dystonic movements, ataxia, and seizures 309530 Mental retardation, X-linked 1, non-dysmorphic 309545 Mental retardation, X-linked nonspecific, with aphasia 309548 Mental retardation, X-linked, FRAXE type 309555 Gustavson syndrome 309585 Mental retardation, X-linked, syndromic-6, with gynecomastia and obesity 309600 Allan-Herndon syndrome 309605 Mental retardation, X-linked, syndromic-4, with congenital contractures and low fingertip arches 309610 Mental retardation, X-linked, syndromic-2, with dysmorphism and cerebral atrophy 309620 Mental retardation-skeletal dysplasia 309850 Brunner syndrome 309900 Mucopolysaccharidosis II 310300 Emery-Dreifuss muscular dystrophy 310400 Myotubular myopathy, X-linked 310460 Myopia-1 310490 Cowchock syndrome 310500 Night blindness, congenital stationary, type 1 311050 Optic atrophy, X-linked 311200 Oral-facial-digital syndrome 1 311250 Ornithine transcarbamylase deficiency 311300 Otopalatodigital syndrome, type I 311360 Ovarian failure, premature 311510 Waisman parkinsonism-mental retardation syndrome 311800 Myoglobinuria/hemolysis due to PGK deficiency 311850 Phosphoribosyl pyrophosphate synthetase-related gout 311870 Muscle glycogenosis 312000 Panhypopituitarism, X-linked 312040 N syndrome, 310465 312060 Properdin deficiency, X-linked 312080 Pelizaeus-Merzbacher disease 312170 Pyruvate dehydrogenase deficiency 312600 Retinitis pigmentosa-2 312610 Retinitis pigmentosa-3 312700 Retinoschisis 312760 Turner syndrome 313350 Split hand/foot malformation, type 2 313400 Spondyloepiphyseal dysplasia tarda 313700 Perineal hypospadias 313850 Thoracoabdominal syndrome 314200 [Euthyroidal hyper- and hypothyroxinemia] 314250 Dystonia-3, torsion, with parkinsonism, Filipino type 314300 Goeminne TKCR syndrome 314400 Cardiac valvular dysplasia-1 314580 Wieacker-Wolff syndrome 314850 McLeod phenotype 600020 Prostate cancer, 176807 600035 Schizencephaly 600040 Colorectal cancer 600044 Thrombocythemia, essential, 187950 600045 Xeroderma pigmentosum, group E, subtype 2 600048 Breast cancer-3 600059 Retinitis pigmentosa-13 600065 Leukocyte adhesion deficiency, 116920 600079 Colon cancer 600095 Split hand/foot malformation, type 3 600101 Deafness, autosomal dominant 2 600105 Retinitis pigmentosa-12, autosomal recessive 600119 Muscular dystrophy, Duchenne-like, type 2 600138 Retinitis pigmentosa-11 600140 Rubenstein-Taybi syndrome, 180849 600143 Epilepsy, progressive, with mental retardation 600151 Bardet-Biedl syndrome 3 600160 Melanoma, 155601 600163 Long QT syndrome-3 600173 SCID, autosomal recessive, T-negative/B-positive type 600175 Spinal muscular atrophy, congenital nonprogressive, of lower limbs 600179 Leber congenital amaurosis, type I, 204000 600184 Carnitine acetyltransferase deficiency 600185 Pancreatic cancer 600194 Ichthyosis bullosa of Siemens, 146800 600202 Dyslexia, specific, 2 600211 Cleidocranial dysplasia, 119600 600221 Venous malformations, multiple cutaneous and mucosal, 600195 600223 Spinocerebellar ataxia-4 600225 Phenylketonuria, atypical, due to GCH1 deficiency, 233910 600228 Pseudohypoaldosteronism, type I, 264350 600231 Palmoplantar keratoderma, Bothnia type 600234 HMG-CoA synthease-2 deficiency 600243 Temperature-sensitive apoptosis 600258 Colorectal cancer, hereditary nonpolyposis, type 3 600259 Turcot syndrome with glioblastoma, 276300 600261 Ehlers-Danlos-like syndrome 600266 Resistance/susceptibility to TB, etc. 600273 Polycystic kidney disease, infantile severe, with tuberous sclerosis 600276 Cerebral arteriopathy with subcortical infarcts and leukoencephalopathy, 125310 600281 Non-insulin-dependent diabetes mellitus, 125853 600309 Atrioventricular canal defect-1 600310 Pseudoachondroplasia, 177170 600318 Diabetes mellitus, insulin-dependent, 3 600319 Diabetes mellitus, insulin-dependent, 4 600320 Insulin-dependent diabetes mellitus-5 600321 Diabetes mellitus, insulin-dependent, 7 600332 Rippling muscle disease-1 600354 Spinal muscular atrophy-1, 253300 600359 Bartter syndrome, type 2 600363 Spastic paraplegia-6 600364 Cone dystrophy-3, 602093 600374 Bardet-Biedl syndrome 4 600414 Adrenoleukodystrophy, neonatal, 202370 600415 Ataxia with isolated vitamin E deficiency, 277460 600429 [Ii blood group, 110800] 600509 Persistent hyperinsulinemic hypoglycemia of infancy, 256450 600510 Pigment dispersion syndrome 600511 Schizophrenia-3 600512 Epilepsy, partial 600525 Trichodontoosseous syndrome, 190320 600528 CPT deficiency, hepatic, type I, 255120 600536 Myopathy, congenital 600542 Chondrosarcoma, extraskeletal myxoid 600584 Atrial septal defect with atrioventricular conduction defects, 108900 600593 Craniosynostosis, Adelaide type 600617 Lipoid adrenal hyperplasia, 201710 600618 Leukemia, acute lymphoblastic 600623 Prostate cancer, 176807 600624 Cone-rod retinal dystrophy-1 600631 Enuresis, nocturnal, 1 600650 Myopathy due to CPT II deficiency, 255110 600652 Deafness, autosomal dominant 4 600669 Epilepsy, generalized, idiopathic 600678 Cancer susceptibility 600698 Salivary adenoma 600700 Lipoma 600701 Lipoma 600722 Ceroid lipofuscinosis, neuronal, variant juvenile type, with granular osmiophilic deposits 600725 Holoprosencephaly-3, 142945 600757 Orofacial cleft-3 600759 Alzheimer disease-4 600760 Pseudohypoaldosteronism, type I, 264350 600761 Pseudohypoaldosteronism, type I, 264350 600792 Deafness, autosomal recessive 5 600795 Dementia, familial, nonspecific 600807 Bronchial asthma 600808 Enuresis, nocturnal, 2 600811 Xeroderma pigmentosum, group E, DDB-negative subtype, 278740 600835 AIDS, resistance to 600837 Hirschsprung disease, 142623 600839 Bartter syndrome, 241200 600850 Schizophrenia disorder-4 600852 Retinitis pigmentosa-17 600856 Beckwith-Wiedemann syndrome, 130650 600881 Cataract, congenital, zonular, with sutural opacities 600882 Charcot-Marie-Tooth neuropathy-2B 600883 Diabetes mellitus, insulin-dependent, 8 600884 Cardiomyopathy, familial dilated 1B 600887 Endometrial carcinoma 600897 Cataract, zonular pulverulent-1, 116200 600900 Muscular dystrophy, limb-girdle, type 2E 600918 Cystinuria, type III 600919 Long QT syndrome-4 with sinus bradycardia 600923 Porphyria variegata, 176200 600937 Persistent hyperinsulinemic hypoglycemia of infancy, 256450 600946 Short stature, autosomal dominant, with normal serum growth hormone binding protein 600956 Persistent Mullerian duct syndrome, type II, 261550 600957 Persistent Mullerian duct syndrome, type I, 261550 600958 Cardiomyopathy, familial hypertrophic, 4, 115197 600965 Deafness, autosomal dominant 6 600968 Gitelman syndrome, 263800 600971 Deafness, autosomal recessive 6 600974 Deafness, autosomal recessive 7 600975 Glaucoma 3, primary infantile, B 600977 Cone dystrophy, progressive 600983 Pseudohypoaldosteronism type I, autosomal dominant, 177735 600993 Pancreatic cancer 600994 Deafness, autosomal dominant 5 600995 Nephrotic syndrome, idiopathic, steroid-resistant 600996 Arrhythmogenic right ventricular dysplasia-2 600998 Bleeding diathesis due to GNAQ deficiency 601002 5-oxoprolinuria, 266130 601011 Spinocerebellar ataxia-6, 183086 601071 Deafness, autosomal recessive 9 601072 Deafness, autosomal recessive 8 601090 Iridogoniodysgenesis, 601631 601097 Neuropathy, recurrent, with pressure palsies, 162500 601105 Pycnodysostosis, 265800 601107 Dubin-Johnson syndrome, 237500 601130 Tolbutamide poor metabolizer 601145 Epilepsy, progressive myoclonic 1, 254800 601146 Brachydactyly, type C, 113100 601154 Cardiomyopathy, dilated, 1E 601199 Neonatal hyperparathyroidism, 239200 601202 Cataract, anterior polar-2 601208 Insulin-dependent diabetes mellitus-11 601226 Progressive external ophthalmoplegia, type 2 601238 Cerebellar ataxia, Cayman type 601267 HIV infection, susceptibility/resistence to 601277 Ichthyosis, lamellar, type 2 601282 Muscular dystrophy with epidermolysis bullosa simplex, 226670 601284 Hereditary hemorrhagic telangiectasia-2, 600376 601295 Bile acid malabsorption, primary 601309 Basal cell carcinoma, sporadic 601313 Polycystic kidney disease, adult type I, 173900 601316 Deafness, autosomal dominant 10 601318 Diabetes mellitus, insulin-dependent, 13 601362 DiGeorge syndrome/velocardiofacial syndrome complex-2 601363 Wilms tumor, type 4 601369 Deafness, autosomal dominant 9 601373 HIV infection, susceptibility/resistance to 601382 Charcot-Marie-Tooth neuropathy-4B 601385 Prostate cancer 601386 Deafness, autosomal recessive 12 601387 Breast cancer 601399 Platelet disorder, familial, with associated myeloid malignancy 601402 Leukemia, myeloid, acute 601406 B-cell non-Hodgkin lymphoma, high-grade 601410 Diabetes mellitus, transient neonatal 601411 Muscular dystrophy, limb-girdle, type 2F, 601287 601412 Deafness, autosomal dominant 7 601414 Retinitis pigmentosa-18 601455 Hereditary motor and sensory neuropathy, Lom type 601458 Inflammatory bowel disease-2 601471 Moebius syndrome-2 601472 Charcot-Marie-Tooth neuropathy-2D 601493 Cardiomyopathy, dilated 1C 601494 Cardiomyopathy, familial, dilated-2 601498 Peroxisomal biogenesis disorder, complementation group 4 601517 Spinocerebellar ataxia-2, 183090 601518 Prostate cancer, hereditary, 1, 176807 601542 Rieger syndrome, type 1, 180500 601545 Lissencephaly-1 601556 Spinocerebellar ataxia-1, 164400 601567 Combined factor V and VIII deficiency, 227300 601596 Charcot-Marie-Tooth neuropathy, demyelinating 601604 Mycobacterial and salmonella infections, susceptibility to 601606 Trichoepithelioma, multiple familial 601607 Rhabdoid tumors 601620 Holt-Oram syndrome, 142900 601621 Ulnar-mammary syndrome, 181450 601622 Saethre-Chotzen syndrome, 101400 601623 Angelman syndrome 601649 Blepharophimosis, epicanthus inversus, and ptosis, type 2 601650 Paraganglioma, familial nonchromaffin, 2 601652 Glaucoma 1A, primary open angle, juvenile-onset, 137750 601653 Branchiootic syndrome 601666 Insulin-dependent diabetes mellitus-15 601669 Hirschsprung disease, one form 601676 Acute insulin response 601680 Distal arthrogryposis, type 2B 601682 Glaucoma 1C, primary open angle 601687 Meesmann corneal dystrophy, 122100 601690 Platelet-activating factor acetylhydrolase deficiency 601691 Retinitis pigmentosa-19, 601718 601692 Reis-Bucklers corneal dystrophy 601718 Retinitis pigmentosa-19 601728 Bannayan-Zonana syndrome, 153480 601744 Systemic lupus erythematosus, susceptibility to, 1 601757 Rhizomelic chondrodysplasia punctata, type 1, 215100 601768 Leukemia, acute myeloid 601769 Osteoporosis, involutional 601771 Glaucoma 3A, primary infantile, 231300 601777 Cone dystrophy, progressive 601780 Ceroid-lipofuscinosis, neuronal-6, variant late infantile 601785 Carbohydrate-deficient glycoprotein syndrome, type I, 212065 601800 [Hair color, brown] 601843 Hypothyroidism, congenital, 274400 601844 Pseudohypoaldosteronism type II 601846 Muscular dystrophy with rimmed vacuoles 601847 Progressive intrahepatic cholestasis-2 601850 Retinitis pigmentosa-deafness syndrome 601863 Bare lymphocyte syndrome, complementation group C 601868 Deafness, autosomal dominant 13 601884 [High bone mass] 601885 Cataract, zonular pulverulent-2 601889 Lymphoma, diffuse large cell 601916 Pancreatic cancer 601920 Alagille syndrome, 118450 601928 Monilethrix, 158000 601941 Insulin-dependent diabetes mellitus-6 601954 Muscular dystrophy, limb-girdle, type 2G 601969 Medulloblastoma, 155255 601975 Ectodermal dysplasia/skin fragility syndrome 601990 Neuroblastoma 602014 Hypomagnesemia with secondary hypocalcemia 602023 Bartter syndrome, type 3 602025 Obesity/hyperinsulinism, susceptibility to 602028 Multiple myeloma 602066 Convulsions, infantile and paroxysmal choreoathetosis 602067 Cardiomyopathy, dilated, 1F 602078 Fibrosis of extraocular muscles, congenital, 2 602080 Paget disease of bone-2 602081 Speech-language disorder-1 602082 Corneal dystrophy, Thiel-Behnke type 602084 Endometrial carcinoma 602085 Postaxial polydactyly, type A2 602086 Arrhythmogenic right ventricular dysplasia-3 602087 Arrhythmogenic right ventricular dysplasia-4 602088 Nephronophthisis, infantile 602089 Hemangioma, capillary, hereditary 602091 Marfan syndrome, atypical 602092 Deafness, autosomal recessive 18 602094 Lipodystrophy, familial partial 602096 Alzheimer disease-5 602099 Amytrophic lateral sclerosis-5 602116 Glioma 602117 Prader-Willi syndrome 602121 Deafness, autosomal dominant nonsyndromic sensorineural, 1, 124900 602134 Tremor, familial essential, 2 602136 Refsum disease, infantile, 266510 602153 Monilethrix, 158000 602216 Peutz-Jeghers syndrome, 175200 602221 Stem-cell leukemia/lymphoma syndrome 602225 Cone-rod retinal dystrophy-2, 120970 602229 Waardenburg-Shah syndrome, 277580 602232 Epilepsy, benign neonatal, type 2, 121201 602235 Epilepsy, benign, neonatal, type 1, 121200 602279 Oculopharyngeal muscular dystorphy, 164300 602280 Retinitis pigmentosa-14, 600132 602363 Ellis-van Creveld-like syndrome 602397 Cholestasis, benign recurrent intrahepatic, 243300 602403 Alzheimer disease, susceptibility to 602404 Parkinson disease, type 3 602421 Sweat chloride elevation without CF 602447 Coronary artery disease, susceptibility to 602460 Deafness, autosomal dominant 15, 602459 602475 Ossification of posterior longitudinal ligament of spine 602476 Febrile convulsions, familial, 1 602477 Febrile convulsions, familial, 2 602491 Hyperlipidemia, familial combined, 1 602522 Bartter syndrome, infantile, with sensorineural deafness 602544 Parkinson disease, juvenile, type 2, 600116 602568 Homocystinuria-megaloblastic anemia, cbl E type, 236270 602574 Deafness, autosomal dominant 12, 601842 602575 Nail-patella syndrome with open-angle glaucoma, 137750 602616 Carbohydrate-deficient glycoprotein syndrome, type II, 212066 602629 Dystonia-6, torsion 602631 Rhabdomyosarcoma, 268210 602666 Deafness, autosomal recessive 3, 600316 602669 Anterior segment mesenchymal dysgenesis and cataract, 107250 602685 Mental retardation, severe, with spasticity and tapetoretinal degeneration 602716 Nephrosis-1, congenital, Finnish type, 256300 602759 Prostate cancer, hereditary, 2, 176807 602771 Muscular dystrophy, congenital, with early spine rigidity 602772 Retinitis pitmentosa-24 602782 Faisalabad histiocytosis 602783 Spastic paraplegia-7

Mature Polypeptides

The present invention also encompasses mature forms of a polypeptide having the amino acid sequence of SEQ ID NO:Y and/or the amino acid sequence encoded by the cDNA in a deposited clone. Polynucleotides encoding the mature forms (such as, for example, the polynucleotide sequence in SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone) are also encompassed by the invention. Moreover, fragments or variants of these polypeptides (such as, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide encoding these polypeptides) are also encompassed by the invention. In preferred embodiments, these fragments or variants retain one or more functional activities of the full-length or mature form of the polypeptide (e.g., biological activity (such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating disorders such as immune, cardiovascular, cancer, and other proliferative disorders), antigenicity (ability to bind, or compete with a polypeptide of the invention for binding, to an anti-polypeptide of the invention antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention). Antibodies that bind the polypeptides of the invention, and polynucleotides encoding these polypeptides are also encompassed by the invention.

According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretary leader sequence that is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein. Further, it has long been known that cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.

Methods for predicting whether a protein has a signal sequence, as well as the cleavage point for that sequence, are available. For instance, the method of McGeoch, Virus Res. 3:271-286 (1985), uses the information from a short N-terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje, Nucleic Acids Res. 14:4683-4690 (1986) uses the information from the residues surrounding the cleavage site, typically residues −13 to +2, where +1 indicates the amino terminus of the secreted protein. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. (von Heinje, supra.) However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.

In the present case, the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called SignalP (Henrik Nielsen et al., Protein Engineering 10:1-6 (1997)), which predicts the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis of the amino acid sequences of the secreted proteins described herein by this program provided the results shown in Table 1A.

In specific embodiments, polypeptides of the invention comprise, or alternatively consist of, the predicted mature form of the polypeptide as delineated in columns 14 and 15 of Table 1A. Moreover, fragments or variants of these polypeptides (such as, fragments as described herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polypeptides, or polypeptides encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of the polynucleotide encoding these polypeptides) are also encompassed by the invention. In preferred embodiments, these fragments or variants retain one or more functional activities of the full-length or mature form of the polypeptide (e.g., biological activity (such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating disorders such as immune, cardiovascular, cancer, and other proliferative disorders), antigenicity (ability to bind, or compete with a polypeptide of the invention for binding, to an anti-polypeptide of the invention antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention). Antibodies that bind the polypeptides of the invention, and polynucleotides encoding these polypeptides are also encompassed by the invention.

Polynucleotides encoding proteins comprising, or consisting of, the predicted mature form of polypeptides of the invention (e.g., polynucleotides having the sequence of SEQ ID NO:X (Table 1A, column 5), the sequence delineated in columns 7 and 8 of Table 1A, and a sequence encoding the mature polypeptide delineated in columns 14 and 15 of Table 1A (e.g., the sequence of SEQ ID NO:X encoding the mature polypeptide delineated in columns 14 and 15 of Table 1A) are also encompassed by the invention, as are fragments or variants of these polynucleotides (such as, fragments as described herein, polynucleotides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to these polynucleotides, and nucleic acids which hybridizes under stringent conditions to the complementary strand of the polynucleotide).

As one of ordinary skill would appreciate, however, cleavage sites sometimes vary from organism to organism and cannot be predicted with absolute certainty. Accordingly, the present invention provides secreted polypeptides having a sequence shown in SEQ ID NO:Y which have an N-terminus beginning within 15 residues of the predicted cleavage point (i.e., having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 more or less contiguous residues of SEQ ID NO:Y at the N-terminus when compared to the predicted mature form of the polypeptide (e.g., the mature polypeptide delineated in columns 14 and 15 of Table 1A). Similarly, it is also recognized that in some cases, cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species. These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence. For example, the naturally occurring signal sequence may be further upstream from the predicted signal sequence. However, it is likely that the predicted signal sequence will be capable of directing the secreted protein to the ER. Nonetheless, the present invention provides the mature protein produced by expression of the polynucleotide sequence of SEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA of a deposited clone, in a mammalian cell (e.g., COS cells, as described below). These polypeptides, and the polynucleotides encoding such polypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention is also directed to variants of the polynucleotide sequence disclosed in SEQ ID NO:X or the complementary strand thereto, nucleotide sequences encoding the polypeptide of SEQ ID NO:Y, the nucleotide sequence of SEQ ID NO:X that encodes the polypeptide sequence as defined in columns 11, 12, 13, 14, and/or 15 of Table 1A, nucleotide sequences encoding the polypeptide sequence as defined in columns 11, 12, 13, 14, and/or 15 of Table 1A, the nucleotide sequence of SEQ ID NO:X encoding the polypeptide sequence as defined in Table 1B.1 (such as the sequence defined in column 5 of Table 1B.1), nucleotide sequences encoding the polypeptide as defined in Table 1B.1 (such as the sequence defined in columns 6 and 7 of Table 1B.1), the nucleotide sequence as defined in columns 8 and 9 of Table 2, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, the nucleotide sequence as defined in column 6 of Table 1C, nucleotide sequences encoding the polypeptide encoded by the nucleotide sequence as defined in column 6 of Table 1C, the cDNA sequence contained in ATCC™ Deposit No:Z, nucleotide sequences encoding the polypeptide encoded by the cDNA sequence contained in ATCC™ Deposit No:Z, and/or nucleotide sequences encoding a mature (secreted) polypeptide encoded by the cDNA sequence contained in

ATCC™ Deposit No:Z.

The present invention also encompasses variants of the polypeptide sequence disclosed in SEQ ID NO:Y, the polypeptide as defined in columns 11, 12, 13, 14, and/or 15 of Table 1A, the polypeptide sequence as defined in Table 1B.1 (such as the sequence defined in columns 6 and 7 of Table 1B.1), a polypeptide sequence encoded by the polynucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2, a polypeptide sequence encoded by the nucleotide sequence as defined in column 6 of Table 1C, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, the polypeptide sequence encoded by the cDNA sequence contained in ATCC™ Deposit No:Z and/or a mature (secreted) polypeptide encoded by the cDNA sequence contained in ATCC™ Deposit No:Z.

“Variant” refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. Generally, variants are overall closely similar, and, in many regions, identical to the polynucleotide or polypeptide of the present invention.

Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence described in SEQ ID NO:X or contained in the cDNA sequence of ATCC™ Deposit No:Z; (b) a nucleotide sequence in SEQ ID NO:X or the cDNA in ATCC™ Deposit No:Z which encodes the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC™ Deposit No:Z; (c) a nucleotide sequence in SEQ ID NO:X or the cDNA in ATCC™ Deposit No:Z which encodes a mature polypeptide (i.e., a secreted polypeptide (e.g., as delineated in columns 14 and 15 of Table 1A)); (d) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of ATCC™ Deposit No:Z, which encodes a biologically active fragment of a polypeptide; (e) a nucleotide sequence in SEQ ID NO:X or the cDNA sequence of ATCC™ Deposit No:Z, which encodes an antigenic fragment of a polypeptide; (f) a nucleotide sequence encoding a polypeptide comprising the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC™ Deposit No:Z; (g) a nucleotide sequence encoding a mature polypeptide of the amino acid sequence of SEQ ID NO:Y (i.e., a secreted polypeptide (e.g., as delineated in columns 14 and 15 of Table 1A)) or a mature polypeptide of the amino acid sequence encoded by the cDNA in ATCC™ Deposit No:Z; (h) a nucleotide sequence encoding a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC™ Deposit No:Z; (i) a nucleotide sequence encoding an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC™ Deposit No:Z; and (j) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i) above.

The present invention is also directed to nucleic acid molecules which comprise, or alternatively consist of, a nucleotide sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), or (j) above, the nucleotide coding sequence in SEQ ID NO:X or the complementary strand thereto, the nucleotide coding sequence of the cDNA contained in ATCC™ Deposit No:Z or the complementary strand thereto, a nucleotide sequence encoding the polypeptide of SEQ ID NO:Y, a nucleotide sequence encoding a polypeptide sequence encoded by the nucleotide sequence in SEQ ID NO:X, a polypeptide sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, a nucleotide sequence encoding the polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z, the nucleotide coding sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto, the nucleotide coding sequence in SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto, a nucleotide sequence encoding the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto, the nucleotide sequence in SEQ ID NO:X encoding the polypeptide sequence as defined in Table 1B.1 (such as the sequence defined in columns 6 and 7 of Table 1B.1) or the complementary strand thereto, nucleotide sequences encoding the polypeptide as defined in Table 1B.1 (such as the sequence defined in columns 6 and 7 of Table 1B.1) or the complementary strand thereto, and/or polynucleotide fragments of any of these nucleic acid molecules (e.g., those fragments described herein). Polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides and nucleic acids.

In a preferred embodiment, the invention encompasses nucleic acid molecules which comprise, or alternatively, consist of a polynucleotide which hybridizes under stringent hybridization conditions, or alternatively, under lower stringency conditions, to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), or (i), above, as are polypeptides encoded by these polynucleotides.

In another preferred embodiment, polynucleotides which hybridize to the complement of these nucleic acid molecules under stringent hybridization conditions, or alternatively, under lower stringency conditions, are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.

In another embodiment, the invention provides a purified protein comprising, or alternatively consisting of, a polypeptide having an amino acid sequence selected from the group consisting of: (a) the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC™ Deposit No:Z; (b) the amino acid sequence of a mature (secreted) form of a polypeptide having the amino acid sequence of SEQ ID NO:Y (e.g., as delineated in columns 14 and 15 of Table 1A) or a mature form of the amino acid sequence encoded by the cDNA in ATCC™ Deposit No:Z mature; (c) the amino acid sequence of a biologically active fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC™ Deposit No:Z; and (d) the amino acid sequence of an antigenic fragment of a polypeptide having the complete amino acid sequence of SEQ ID NO:Y or the complete amino acid sequence encoded by the cDNA in ATCC™ Deposit No:Z.

The present invention is also directed to proteins which comprise, or alternatively consist of, an amino acid sequence which is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, identical to, for example, any of the amino acid sequences in (a), (b), (c), or (d), above, the amino acid sequence shown in SEQ ID NO:Y, the amino acid sequence encoded by the cDNA contained in ATCC™ Deposit No:Z, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X as defined in columns 8 and 9 of Table 2, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C, the amino acid sequence as defined in Table 1B.1 (such as the sequence defined in columns 6 and 7 of Table 1B.1), an amino acid sequence encoded by the nucleotide sequence in SEQ ID NO:X, and an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X. Fragments of these polypeptides are also provided (e.g., those fragments described herein). Further proteins encoded by polynucleotides which hybridize to the complement of the nucleic acid molecules encoding these amino acid sequences under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention, as are the polynucleotides encoding these proteins.

By a nucleic acid having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be an entire sequence referred to in Tables 1B.1 or 2 as the ORF (open reading frame), or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a query amino acid sequence of the present invention, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, (indels) or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence of a polypeptide referred to in Table 1A (e.g., the amino acid sequence delineated in columns 14 and 15) or a fragment thereof, Table 1B.1 (e.g., the amino acid sequence identified in column 6) or a fragment thereof, Table 2 (e.g., the amino acid sequence of the polypeptide encoded by the polynucleotide sequence defined in columns 8 and 9 of Table 2) or a fragment thereof, the amino acid sequence of the polypeptide encoded by the polynucleotide sequence in SEQ ID NO:B as defined in column 6 of Table 1C or a fragment thereof, the amino acid sequence of the polypeptide encoded by the nucleotide sequence in SEQ ID NO:X or a fragment thereof, or the amino acid sequence of the polypeptide encoded by cDNA contained in ATCC™ Deposit No:Z, or a fragment thereof, the amino acid sequence of a mature (secreted) polypeptide encoded by cDNA contained in ATCC™ Deposit No:Z, or a fragment thereof, can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is expressed as percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to made for the purposes of the present invention.

The polynucleotide variants of the invention may contain alterations in the coding regions, non-coding regions, or both. Especially preferred are polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide. Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred. Moreover, polypeptide variants in which less than 50, less than 40, less than 30, less than 20, less than 10, or 5-50, 5-25, 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred. Polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those preferred by a bacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNA technology, variants may be generated to improve or alter the characteristics of the polypeptides of the present invention. For instance, one or more amino acids can be deleted from the N-terminus or C-terminus of the polypeptide of the present invention without substantial loss of biological function. As an example, Ron et al. (J. Biol. Chem. 268: 2984-2988 (1993)) reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216 (1988).)

Moreover, ample evidence demonstrates that variants often retain a biological activity similar to that of the naturally occurring protein. For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993)) conducted extensive mutational analysis of human cytokine IL-1a. They used random mutagenesis to generate over 3,500 individual IL-1a mutants that averaged 2.5 amino acid changes per variant over the entire length of the molecule. Multiple mutations were examined at every possible amino acid position. The investigators found that “[m]ost of the molecule could be altered with little effect on either [binding or biological activity].” In fact, only 23 unique amino acid sequences, out of more than 3,500 nucleotide sequences examined, produced a protein that significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from the N-terminus or C-terminus of a polypeptide results in modification or loss of one or more biological functions, other biological activities may still be retained. For example, the ability of a deletion variant to induce and/or to bind antibodies which recognize the secreted form will likely be retained when less than the majority of the residues of the secreted form are removed from the N-terminus or C-terminus. Whether a particular polypeptide lacking N- or C-terminal residues of a protein retains such immunogenic activities can readily be determined by routine methods described herein and otherwise known in the art.

Thus, the invention further includes polypeptide variants which show a biological or functional activity of the polypeptides of the invention (such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating disorders such as immune or cardiovascular disorders). Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.

The present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, (e.g., encoding a polypeptide having the amino acid sequence of an N and/or C terminal deletion), irrespective of whether they encode a polypeptide having functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having functional activity include, inter alia, (1) isolating a gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphase chromosomal spreads to provide precise chromosomal location of the gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); (3) Northern Blot analysis for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues); and (4) in situ hybridization (e.g., histochemistry) for detecting mRNA expression in specific tissues (e.g., normal or diseased tissues).

Preferred, however, are nucleic acid molecules having sequences at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequences disclosed herein, which do, in fact, encode a polypeptide having functional activity. By a polypeptide having “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) protein and/or a mature (secreted) protein of the invention. Such functional activities include, but are not limited to, biological activity (such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders such as immune, cardiovascular, cancer, and other proliferative diseases and disorders), antigenicity (ability to bind, or compete with a polypeptide of the invention for binding, to an anti-polypeptide of the invention antibody), immunogenicity (ability to generate antibody which binds to a specific polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention.

The functional activity of the polypeptides, and fragments, variants, derivatives, and analogs of the invention, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability to bind or compete with a full-length polypeptide of the present invention for binding to an anti-polypetide antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

In another embodiment, where a ligand is identified, or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, the ability of physiological correlates of a polypeptide of the present invention to bind to a substrate(s) of the polypeptide of the invention can be routinely assayed using techniques known in the art.

In addition, assays described herein (see Examples) and otherwise known in the art may routinely be applied to measure the ability of polypeptides of the present invention and fragments, variants, derivatives, and analogs thereof to elicit polypeptide related biological activity (either in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention.

Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to, for example, the nucleic acid sequence of the cDNA contained in ATCC™ Deposit No:Z, the nucleic acid sequence referred to in Tables 1B.1 and 1B.2 (SEQ ID NO:X), the nucleic acid sequence disclosed in Table 1A (e.g., the nucleic acid sequence delineated in columns 7 and 8), the nucleic acid sequence disclosed in Table 2 (e.g., the nucleic acid sequence delineated in columns 8 and 9) or fragments thereof, will encode polypeptides “having functional activity.” In fact, since degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having functional activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.

For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.

The second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanine-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. See Cunningham and Wells, Science 244:1081-1085 (1989). The resulting mutant molecules can then be tested for biological activity.

As the authors state, these two strategies have revealed that proteins are surprisingly tolerant of amino acid substitutions. The authors further indicate which amino acid changes are likely to be permissive at certain amino acid positions in the protein. For example, most buried (within the tertiary structure of the protein) amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Moreover, tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gln, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

Besides conservative amino acid substitution, variants of the present invention include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitutions with one or more of the amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, serum albumin (preferably human serum albumin) or a fragment thereof, or leader or secretory sequence, or a sequence facilitating purification, or (v) fusion of the polypeptide with another compound, such as albumin (including but not limited to recombinant albumin (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity. See Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993).

A further embodiment of the invention relates to polypeptides which comprise the amino acid sequence of a polypeptide having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions from a polypeptide sequence disclosed herein. Of course it is highly preferable for a polypeptide to have an amino acid sequence which, for example, comprises the amino acid sequence of a polypeptide of SEQ ID NO:Y, the amino acid sequence of the mature (e.g., secreted) polypeptide of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X, an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, an amino acid sequence encoded by the complement of SEQ ID NO:X, an amino acid sequence encoded by cDNA contained in ATCC™ Deposit No:Z, and/or the amino acid sequence of a mature (secreted) polypeptide encoded by cDNA contained in ATCC™ Deposit No:Z, or a fragment thereof, which contains, in order of ever-increasing preference, at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.

In specific embodiments, the polypeptides of the invention comprise, or alternatively, consist of, fragments or variants of a reference amino acid sequence selected from: (a) the amino acid sequence of SEQ ID NO:Y or fragments thereof (e.g., the mature form and/or other fragments described herein); (b) the amino acid sequence encoded by SEQ ID NO:X or fragments thereof; (c) the amino acid sequence encoded by the complement of SEQ ID NO:X or fragments thereof; (d) the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or fragments thereof; and (e) the amino acid sequence encoded by cDNA contained in ATCC™ Deposit No:Z or fragments thereof; wherein the fragments or variants have I-5, 5-10, 5-25, 5-50, 10-50 or 50-150, amino acid residue additions, substitutions, and/or deletions when compared to the reference amino acid sequence. In preferred embodiments, the amino acid substitutions are conservative. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Polynucleotide and Polypeptide Fragments

The present invention is also directed to polynucleotide fragments of the polynucleotides (nucleic acids) of the invention. In the present invention, a “polynucleotide fragment” refers to a polynucleotide having a nucleic acid sequence which, for example: is a portion of the cDNA contained in ATCC™ Deposit No:Z or the complementary strand thereto; is a portion of the polynucleotide sequence encoding the polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z or the complementary strand thereto; is a portion of the polynucleotide sequence encoding the mature (secreted) polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the mature amino acid sequence as defined in columns 14 and 15 of Table 1A or the complementary strand thereto; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence of SEQ ID NO:X as defined in columns 8 and 9 of Table 2 or the complementary strand thereto; is a portion of the polynucleotide sequence in SEQ ID NO:X or the complementary strand thereto; is a polynucleotide sequence encoding a portion of the polypeptide of SEQ ID NO:Y; is a polynucleotide sequence encoding a portion of a polypeptide encoded by SEQ ID NO:X; is a polynucleotide sequence encoding a portion of a polypeptide encoded by the complement of the polynucleotide sequence in SEQ ID NO:X; is a portion of a polynucleotide sequence encoding the amino acid sequence encoded by the region of SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto; or is a portion of the polynucleotide sequence of SEQ ID NO:B as defined in column 6 of Table 1C or the complementary strand thereto.

The polynucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length. A fragment “at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in ATCC™ Deposit No:Z, or the nucleotide sequence shown in SEQ ID NO:X or the complementary stand thereto. In this context “about” includes the particularly recited value or a value larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. These nucleotide fragments have uses that include, but are not limited to, as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., at least 160, 170, 180, 190, 200, 250, 500, 600, 1000, or 2000 nucleotides in length) are also encompassed by the invention.

Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of SEQ ID NO:X, or the complementary strand thereto. In this context “about” includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity; such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders such as immune, cardiovascular, cancer, and other proliferative diseases and disorders). More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.

Further representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a sequence from about nucleotide number 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, 2001-2050, 2051-2100, 2101-2150, 2151-2200, 2201-2250, 2251-2300, 2301-2350, 2351-2400, 2401-2450, 2451-2500, 2501-2550, 2551-2600, 2601-2650, 2651-2700, 2701-2750, 2751-2800, 2801-2850, 2851-2900, 2901-2950, 2951-3000, 3001-3050, 3051-3100, 3101-3150, 3151-3200, 3201-3250, 3251-3300, 3301-3350, 3351-3400, 3401-3450, 3451-3500, 3501-3550, 3551-3600, 3601-3650, 3651-3700, 3701-3750, 3751-3800, 3801-3850, 3851-3900, 3901-3950, 3951-4000, 4001-4050, 4051-4100, 4101-4150, 4151-4200, 4201-4250, 4251-4300, 4301-4350, 4351-4400, 4401-4450, 4451-4500, 4501-4550, 4551-4600, 4601-4650, 4651-4700, 4701-4750, 4751-4800, 4801-4850, 4851-4900, 4901-4950, 4951-5000, 5001-5050, 5051-5100, 5101-5150, 5151-5200, 5201-5250, 5251-5300, 5301-5350, 5351-5400, 5401-5450, 5451-5500, 5501-5550, 5551-5600, 5601-5650, 5651-5700, 5701-5750, 5751-5800, 5801-5850, 5851-5900, 5901-5950, 5951-6000, 6001-6050, 6051-6100, 6101-6150, 6151-6200, 6201-6250, 6251-6300, 6301-6350, 6351-6400, 6401-6450, 6451-6500, 6501-6550, 6551-6600, 6601-6650, 6651-6700, 6701-6750, 6751-6800, 6801-6850, 6851-6900, 6901-6950, 6951-7000, 7001-7050, 7051-7100, 7101-7150, 7151-7200, 7201-7250, 7251-7300 or 7301 to the end of the cDNA sequence contained in ATCC™ Deposit No:Z, or the complementary strand thereto. In this context “about” includes the particularly recited range or a range larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Preferably, these fragments encode a polypeptide which has a functional activity (e.g., biological activity). More preferably, these polynucleotides can be used as probes or primers as discussed herein. Polynucleotides which hybridize to one or more of these polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.

Moreover, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence delineated in Table 1C, column 6. Additional, representative examples of polynucleotide fragments of the invention comprise, or alternatively consist of, a nucleic acid sequence comprising one, two, three, four, five, six, seven, eight, nine, ten, or more of the above described polynucleotide fragments of the invention in combination with a polynucleotide sequence that is the complementary strand of a sequence delineated in column 6 of Table 1C. In further embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that of the BAC fragment having the sequence disclosed in SEQ ID NO:B (see Table 1C, column 5). In additional embodiments, the above-described polynucleotide fragments of the invention comprise, or alternatively consist of, sequences delineated in Table 1C, column 6, and have a nucleic acid sequence which is different from that published for the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). In additional embodiments, the above-described polynucleotides of the invention comprise, or alternatively consist of, sequences delineated Table 1C, column 6, and have a nucleic acid sequence which is different from that contained in the BAC clone identified as BAC ID NO:A (see Table 1C, column 4). Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides and polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1C, column 2) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in column 6 of Table 1C which correspond to the same ATCC™ Deposit No:Z (see Table 1C, column 1), and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B, or 1C) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.

In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of, one, two, three, four, five, six, seven, eight, nine, ten, or more fragments of the sequences delineated in the same row of column 6 of Table 1C, and the polynucleotide sequence of SEQ ID NO:X (e.g., as defined in Table 1A, 1B, or 1C) or fragments or variants thereof. Polypeptides encoded by these polynucleotides, other polynucleotides that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of the sequence of SEQ ID NO:X are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids that encode these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.

In additional specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X (e.g., as described herein) are directly contiguous Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.

In further specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of a fragment or variant of the sequence of SEQ ID NO:X and the 5′ 10 polynucleotides of the sequence of one of the sequences delineated in column 6 of Table 1C are directly contiguous. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.

In specific embodiments, polynucleotides of the invention comprise, or alternatively consist of a polynucleotide sequence in which the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C and the 5′ 10 polynucleotides of another sequence in column 6 are directly contiguous. In preferred embodiments, the 3′ 10 polynucleotides of one of the sequences delineated in column 6 of Table 1C is directly contiguous with the 5′ 10 polynucleotides of the next sequential exon delineated in Table 1C, column 6. Nucleic acids which hybridize to the complement of these 20 contiguous polynucleotides under stringent hybridization conditions or alternatively, under lower stringency conditions, are also encompassed by the invention. Polypeptides encoded by these polynucleotides and/or nucleic acids, other polynucleotides and/or nucleic acids encoding these polypeptides, and antibodies that bind these polypeptides are also encompassed by the invention. Additionally, fragments and variants of the above-described polynucleotides, nucleic acids, and polypeptides are also encompassed by the invention.

In the present invention, a “polypeptide fragment” refers to an amino acid sequence which is a portion of the amino acid sequence contained in SEQ ID NO:Y, is a portion of the mature form of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, a portion of an amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, is a portion of an amino acid sequence encoded by the polynucleotide sequence of SEQ ID NO:X, is a portion of an amino acid sequence encoded by the complement of the polynucleotide sequence in SEQ ID NO:X, is a portion of the amino acid sequence of a mature (secreted) polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z, and/or is a portion of an amino acid sequence encoded by the cDNA contained in ATCC™ Deposit No:Z. Protein (polypeptide) fragments may be “free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of cDNA and SEQ ID NO:Y. In a preferred embodiment, polypeptide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, 121-140, 141-160, 161-180, 181-200, 201-220, 221-240, 241-260, 261-280, 281-300, 301-320, 321-340, 341-360, 361-380, 381-400, 401-420, 421-440, 441-460, 461-480, 481-500, 501-520, 521-540, 541-560, 561-580, 581-600, 601-620, 621-640, 641-660, 661-680, 681-700, 701-720, 721-740, 741-760, 761-780, 781-800, 801-820, 821-840, 841-860, 861-880, 881-900, 901-920, 921-940, 941-960, 961-980, 981-1000, 1001-1020, 1021-1040, 1041-1060, 1061-1080, 1081-1100, 1101-1120, 1121-1140, 1141-1160, 1161-1180, 1181-1200, 1201-1220, 1221-1240, 1241-1260, 1261-1280, 1281-1300, 1301-1320, 1321-1340, 1341-1360, 1361-1380, 1381-1400, 1401-1420, 1421-1440, or 1441 to the end of the coding region of SEQ ID NO:Y. Moreover, polypeptide fragments of the invention may be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, or 150 amino acids in length. In this context “about” includes the particularly recited ranges or values, or ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.

Even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities; such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders such as immune, cardiovascular, cancer, and other proliferative diseases and disorders; ability to multimerize; ability to bind a ligand; antigenic ability useful for production of polypeptide specific antibodies) may still be retained. For example, the ability of shortened muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

Accordingly, polypeptide fragments include the secreted protein as well as the mature form. Further preferred polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted polypeptide or the mature form. Similarly, any number of amino acids, ranging from 1-30, can be deleted from the carboxy terminus of the secreted protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are preferred. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.

The present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, a polypeptide as defined in columns 14 and 15 of Table 1A, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X or the complement thereof, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1C, a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z, and/or a mature polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z). In particular, N-terminal deletions may be described by the general formula m-q, where q is a whole integer representing the total number of amino acid residues in a polypeptide of the invention (e.g., the polypeptide disclosed in SEQ ID NO:Y, the mature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, or the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), and m is defined as any integer ranging from 2 to q-6. Polynucleotides encoding these polypeptides are also encompassed by the invention.

The present invention further provides polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of a polypeptide disclosed herein (e.g., a polypeptide of SEQ ID NO:Y, the mature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, a polypeptide encoded by the polynucleotide sequence contained in SEQ ID NO:X, a polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, a polypeptide encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1C, a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z, and/or a mature polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z). In particular, C-terminal deletions may be described by the general formula 1-n, where n is any whole integer ranging from 6 to q-1, and where n corresponds to the position of amino acid residue in a polypeptide of the invention. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In addition, any of the above described N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted polypeptide. The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of a polypeptide encoded by SEQ ID NO:X (e.g., including, but not limited to, the preferred polypeptide disclosed as SEQ ID NO:Y, the mature (secreted) portion of SEQ ID NO:Y as defined in columns 14 and 15 of Table 1A, and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2), the cDNA contained in ATCC™ Deposit No:Z, and/or the complement thereof, where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders such as immune, cardiovascular, cancer, and other proliferative diseases and disorders; ability to multimerize; ability to bind a ligand; antigenic ability useful for production of polypeptide specific antibodies) may still be retained. For example the ability of the shortened mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six amino acid residues may often evoke an immune response.

The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence set forth herein. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific N- and C-terminal deletions. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Any polypeptide sequence encoded by, for example, the polynucleotide sequences set forth as SEQ ID NO:X or the complement thereof, (presented, for example, in Tables 1A and 2), the cDNA contained in ATCC™ Deposit No:Z, or the polynucleotide sequence as defined in column 6 of Table 1C, may be analyzed to determine certain preferred regions of the polypeptide. For example, the amino acid sequence of a polypeptide encoded by a polynucleotide sequence of SEQ ID NO:X (e.g., the polypeptide of SEQ ID NO:Y and the polypeptide encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2) or the cDNA contained in ATCC™ Deposit No:Z may be analyzed using the default parameters of the DNASTAR computer algorithm (DNASTAR, Inc., 1228 S. Park St., Madison, Wis. 53715 USA, world wide web at dnastar.com/).

Also preferred are polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions. Polypeptide regions that may be routinely obtained using the DNASTAR computer algorithm include, but are not limited to, Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle hydrophilic regions and hydrophobic regions; Eisenberg alpha- and beta-amphipathic regions; Karplus-Schulz flexible regions; Emini surface-forming regions; and Jameson-Wolf regions of high antigenic index. Among highly preferred polynucleotides of the invention in this regard are those that encode polypeptides comprising regions that combine several structural features, such as several (e.g., 1, 2, 3 or 4) of the features set out above. Polypeptide fragments of SEQ ID NO:Y falling within conserved domains are specifically contemplated by the present invention. Moreover, polynucleotides encoding these domains are also contemplated.

Additionally, Kyte-Doolittle hydrophilic regions and hydrophobic regions, Emini surface-forming regions, and Jameson-Wolf regions of high antigenic index (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) can routinely be used to determine polypeptide regions that exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from data by DNASTAR analysis by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Preferred polypeptide fragments of the invention are fragments comprising, or alternatively, consisting of, an amino acid sequence that displays a functional activity (e.g. biological activity such as, for example, activity useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders such as immune, cardiovascular, cancer, and other proliferative diseases and disorders; ability to multimerize; ability to bind a ligand; antigenic ability useful for production of polypeptide specific antibodies) of the polypeptide sequence of which the amino acid sequence is a fragment. By a polypeptide displaying a “functional activity” is meant a polypeptide capable of one or more known functional activities associated with a full-length protein, such as, for example, biological activity, antigenicity, immunogenicity, and/or multimerization, as described herein.

Other preferred polypeptide fragments are biologically active fragments. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity. Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.

Preferably, the polynucleotide fragments of the invention encode a polypeptide which demonstrates a functional activity. By a polypeptide demonstrating a “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) polypeptide of invention protein. Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a polypeptide of the invention for binding) to an antibody to the polypeptide of the invention], immunogenicity (ability to generate antibody which binds to a polypeptide of the invention), ability to form multimers with polypeptides of the invention, and ability to bind to a receptor or ligand for a polypeptide of the invention.

The functional activity of polypeptides of the invention, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.

In preferred embodiments, polypeptides of the invention comprise, or alternatively consist of, one, two, three, four, five or more of the antigenic fragments of the polypeptide of SEQ ID NO:Y, or portions thereof. Polynucleotides encoding these polypeptides are also encompassed by the invention.

Epitopes and Antibodies

The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of: the polypeptide sequence shown in SEQ ID NO:Y; a polypeptide sequence encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2; the polypeptide sequence encoded by the portion of SEQ ID NO:B as defined in column 6 of Table 1C or the complement thereto; the polypeptide sequence encoded by the cDNA contained in ATCC™ Deposit No:Z; or the polypeptide sequence encoded by a polynucleotide that hybridizes to the sequence of SEQ ID NO:X, the complement of the sequence of SEQ ID NO:X, the complement of a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, or the cDNA sequence contained in ATCC™ Deposit No:Z under stringent hybridization conditions or alternatively, under lower stringency hybridization as defined supra. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:X, or a fragment thereof), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or alternatively, under lower stringency hybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985) further described in U.S. Pat. No. 4,631,211.)

In the present invention, antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).

Non-limiting examples of epitopes of polypeptides that can be used to generate antibodies of the invention include a polypeptide comprising, or alternatively consisting of, at least one, two, three, four, five, six or more of the portion(s) of SEQ ID NO:Y specified in Table 1B.1 (such as the sequence specified in column 6 of Table 1B.1). These polypeptide fragments have been determined to bear antigenic epitopes of the proteins of the invention by the analysis of the Jameson-Wolf antigenic index that is included in the DNAStar suite of computer programs. By “comprise” it is intended that a polypeptide contains at least one, two, three, four, five, six or more of the portion(s) of SEQ ID NO:Y shown in Table 1B.1 (such as the sequence specified in column 6 of Table 1B.1), but it may contain additional flanking residues on either the amino or carboxyl termini of the recited portion. Such additional flanking sequences are preferably sequences naturally found adjacent to the portion; i.e., contiguous sequence shown in SEQ ID NO:Y. The flanking sequence may, however, be sequences from a heterologous polypeptide, such as from another protein described herein or from a heterologous polypeptide not described herein. In particular embodiments, epitope portions of a polypeptide of the invention comprise one, two, three, or more of the portions of SEQ ID NO:Y shown in Table 1B.1 (such as the sequence specified in column 6 of Table 1B.1).

Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. (Wahl et al., J. Nucl. Med. 24:316-325 (1983).) Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.

Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 μg of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention (e.g., those comprising an immunogenic or antigenic epitope) can be fused to heterologous polypeptide sequences. For example, polypeptides of the present invention (including fragments or variants thereof), may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof, resulting in chimeric polypeptides. By way of another non-limiting example, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)). In a preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322 094) which is herein incorporated by reference in its entirety. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-x of human serum albumin, where x is an integer from 1 to 585 and the albumin fragment has human serum albumin activity. In another preferred embodiment, polypeptides and/or antibodies of the present invention (including fragments or variants thereof) are fused with polypeptide fragments comprising, or alternatively consisting of, amino acid residues 1-z of human serum albumin, where z is an integer from 369 to 419, as described in U.S. Pat. No. 5,766,883 herein incorporated by reference in its entirety. Polypeptides and/or antibodies of the present invention (including fragments or variants thereof) may be fused to either the N- or C-terminal end of the heterologous protein (e.g., immunoglobulin Fc polypeptide or human serum albumin polypeptide). Polynucleotides encoding fusion proteins of the invention are also encompassed by the invention.

Such fusion proteins as those described above may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813). IgG fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (HA) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusion proteins. For example, the polypeptide of the present invention, when fused to a second protein, can be used as an antigenic tag. Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide. Moreover, because secreted proteins target cellular locations based on trafficking signals, polypeptides of the present invention which are shown to be secreted can be used as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but may occur through linker sequences.

In certain preferred embodiments, proteins of the invention are fusion proteins comprising an amino acid sequence that is an N and/or C-terminal deletion of a polypeptide of the invention. In preferred embodiments, the invention is directed to a fusion protein comprising an amino acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to a polypeptide sequence of the invention. Polynucleotides encoding these proteins are also encompassed by the invention.

Moreover, fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.

As one of skill in the art will appreciate that, as discussed above, polypeptides of the present invention, and epitope-bearing fragments thereof, can be combined with heterologous polypeptide sequences. For example, the polypeptides of the present invention may be fused with heterologous polypeptide sequences, for example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), or albumin (including, but not limited to, native or recombinant human albumin or fragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life in vivo. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86 (1988).) Fusion proteins having disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).) For example, EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (EP-A 0232 262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5 See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995). Polynucleotides comprising or alternatively consisting of nucleic acids which encode these fusion proteins are also encompassed by the invention.

Moreover, the polypeptides of the present invention can be fused to marker sequences, such as a polypeptide which facilitates purification of the fused polypeptide. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Another peptide tag useful for purification, the “HA” tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)).

Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:X and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Thus, any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.

Recombinant and Synthetic Production of Polypeptides of the Invention

The present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by synthetic and recombinant techniques. The vector may be, for example, a phage, plasmid, viral, or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

The polynucleotides of the invention may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase, G418, glutamine synthase, or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC™ Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBLUESCRIPT™ vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from STRATAGENE™ Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from PHARIVIACIA™ Biotech, Inc. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from STRATAGENE™; and pSVK3, pBPV, pMSG and pSVL available from PHARMACIA™. Preferred expression vectors for use in yeast systems include, but are not limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlbad, Calif.). Other suitable vectors will be readily apparent to the skilled artisan.

Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors is the availability of cell lines (e.g., the murine myeloma cell line, NS0) which are glutamine synthase negative. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g., Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657, which are hereby incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors can be obtained from Lonza Biologics, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169 (1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are herein incorporated by reference.

The present invention also relates to host cells containing the above-described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art. The host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. A host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled. Furthermore, different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.

Introduction of the nucleic acids and nucleic acid constructs of the invention into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761, issued Mar. 31, 1998; International Publication Number WO 96/29411; International Publication Number WO 94/12650; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. Thus, it is well known in the art that the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express polypeptides of the invention in a eukaryotic system. Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source. A main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O₂. This reaction is catalyzed by the enzyme alcohol oxidase. In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O₂. Consequently, in a growth medium depending on methanol as a main carbon source, the promoter region of one of the two alcohol oxidase genes (AOX1) is highly active. In the presence of methanol, alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987). Thus, a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, under the transcriptional regulation of all or part of the AOX1 regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNA encoding a polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in “Pichia Protocols: Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. The Humana Press, Totowa, N.J., 1998. This expression vector allows expression and secretion of a polypeptide of the invention by virtue of the strong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as, pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct provides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an in-frame AUG as required.

In another embodiment, high-level expression of a heterologous coding sequence, such as, for example, a polynucleotide of the present invention, may be achieved by cloning the heterologous polynucleotide of the invention into an expression vector such as, for example, pGAPZ or pGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

In addition, polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., Nature, 310:105-111 (1984)). For example, a polypeptide corresponding to a fragment of a polypeptide can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides of the present invention which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.

Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include iodine (¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹¹In, ¹¹²In, ^(113m)In, ^(115m)In), technetium (⁹⁹Tc, ^(99m)Tc), thallium gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁴²Pr, ¹⁰⁵Rh, and ⁹⁷Ru.

In specific embodiments, a polypeptide of the present invention or fragment or variant thereof is attached to macrocyclic chelators that associate with radiometal ions, including but not limited to, ¹⁷⁷Lu ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators is ¹¹¹In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator is ⁹⁰Y. In specific embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). In other specific embodiments, DOTA is attached to an antibody of the invention or fragment thereof via a linker molecule. Examples of linker molecules useful for conjugating DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90 (1998); Peterson et al., Bioconjug. Chem. 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50 (1999); which are hereby incorporated by reference in their entirety.

As mentioned, the proteins of the invention may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Polypeptides of the invention may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990); Rattan et al., Ann. N.Y. Acad. Sci. 663:48-62 (1992)).

Also provided by the invention are chemically modified derivatives of the polypeptides of the invention which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, such as, for example, the method disclosed in EP 0 401 384 (coupling PEG to G-CSF), herein incorporated by reference; see also Malik et al., Exp. Hematol. 20:1028-1035 (1992), reporting pegylation of GM-CSF using tresyl chloride. For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation that exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number of additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in International Publication No. WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.

The number of polyethylene glycol moieties attached to each protein of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

The polypeptides of the invention can be recovered and purified from chemical synthesis and recombinant cell cultures by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.

The polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer refers to a multimer containing only polypeptides corresponding to a protein of the invention (e.g., the amino acid sequence of SEQ ID NO:Y, an amino acid sequence encoded by SEQ ID NO:X or the complement of SEQ ID NO:X, the amino acid sequence encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or an amino acid sequence encoded by cDNA contained in ATCC™ Deposit No:Z (including fragments, variants, splice variants, and fusion proteins, corresponding to these as described herein)). These homomers may contain polypeptides having identical or different amino acid sequences. In a specific embodiment, a homomer of the invention is a multimer containing only polypeptides having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptides having different amino acid sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing two polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing three polypeptides having identical and/or different amino acid sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the polypeptides of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked by, for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ ID NO:Y, encoded by the portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or encoded by the cDNA contained in ATCC™ Deposit No:Z). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, osteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the invention involves use of polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.

Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric polypeptides of the invention.

In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in fusion proteins of the invention containing Flag® polypeptide sequence. In a further embodiment, proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag® fusion proteins of the invention and anti-Flag® antibody.

The multimers of the invention may be generated using chemical techniques known in the art. For example, polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hydrophobic or signal peptide) and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

Antibodies

Further polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of the invention (e.g., a polypeptide or fragment or variant of the amino acid sequence of SEQ ID NO:Y or a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z, and/or an epitope, of the present invention) as determined by immunoassays well known in the art for assaying specific antibody-antigen binding. Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In preferred embodiments, the immunoglobulin molecules of the invention are IgG1. In other preferred embodiments, the immunoglobulin molecules of the invention are IgG4.

Most preferably the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).

Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, or by size in contiguous amino acid residues, or listed in the Tables and Figures. Preferred epitopes of the invention include the predicted epitopes shown in Table 1B.1 (such as epitopes shown in column 7 of Table 1B.1), as well as polynucleotides that encode these epitopes. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In a specific embodiment, the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein. Further included in the present invention are antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Preferably, antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have utility in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); incorporated by reference herein in its entirety.

As discussed in more detail below, the antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387; the disclosures of which are incorporated herein by reference in their entireties.

The antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

The antibodies of the present invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples. In a non-limiting example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC™. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.

Another well known method for producing both polyclonal and monoclonal human B cell lines is transformation using Epstein Barr Virus (EBV). Protocols for generating EBV-transformed B cell lines are commonly known in the art, such as, for example, the protocol outlined in Chapter 7.22 of Current Protocols in Immunology, Coligan et al., Eds., 1994, John Wiley & Sons, NY, which is hereby incorporated in its entirety by reference. The source of B cells for transformation is commonly human peripheral blood, but B cells for transformation may also be derived from other sources including, but not limited to, lymph nodes, tonsil, spleen, tumor tissue, and infected tissues. Tissues are generally made into single cell suspensions prior to EBV transformation. Additionally, steps may be taken to either physically remove or inactivate T cells (e.g., by treatment with cyclosporin A) in B cell-containing samples, because T cells from individuals seropositive for anti-EBV antibodies can suppress B cell immortalization by EBV.

In general, the sample containing human B cells is innoculated with EBV, and cultured for 3-4 weeks. A typical source of EBV is the culture supernatant of the B95-8 cell line (ATCC™ #VR-1492). Physical signs of EBV transformation can generally be seen towards the end of the 3-4 week culture period. By phase-contrast microscopy, transformed cells may appear large, clear, hairy and tend to aggregate in tight clusters of cells. Initially, EBV lines are generally polyclonal. However, over prolonged periods of cell cultures, EBV lines may become monoclonal or polyclonal as a result of the selective outgrowth of particular B cell clones. Alternatively, polyclonal EBV transformed lines may be subcloned (e.g., by limiting dilution culture) or fused with a suitable fusion partner and plated at limiting dilution to obtain monoclonal B cell lines. Suitable fusion partners for EBV transformed cell lines include mouse myeloma cell lines (e.g., SP2/0, X63-Ag8.653), heteromyeloma cell lines (human×mouse; e.g, SPAM-8, SBC-H20, and CB-F7), and human cell lines (e.g., GM 1500, SKO-007, RPMI 8226, and KR-4). Thus, the present invention also provides a method of generating polyclonal or monoclonal human antibodies against polypeptides of the invention or fragments thereof, comprising EBV-transformation of human B cells.

Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; 5,939,598; 6,075,181; and 6,114,598, which are incorporated by reference herein in their entirety. In addition, companies such as ABGENIX™, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).

Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that “mimic” the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby block its biological activity. Alternatively, antibodies which bind to and enhance polypeptide multimerization and/or binding, and/or receptor/ligand multimerization, binding and/or signaling can be used to generate anti-idiotypes that function as agonists of a polypeptide of the invention and/or its ligand/receptor. Such agonistic anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens as agonists of the polypeptides of the invention or its ligand(s)/receptor(s). For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligand(s)/receptor(s), and thereby promote or enhance its biological activity.

Intrabodies of the invention can be produced using methods known in the art, such as those disclosed and reviewed in Chen et al., Hum. Gene Ther. 5:595-601 (1994); Marasco, W. A., Gene Ther. 4:11-15 (1997); Rondon and Marasco, Annu Rev. Microbiol. 51:257-283 (1997); Proba et al., J. Mol. Biol. 275:245-253 (1998); Cohen et al., Oncogene 17:2445-2456 (1998); Ohage and Steipe, J. Mol. Biol. 291:1119-1128 (1999); Ohage et al., J. Mol. Biol. 291:1129-1134 (1999); Wirtz and Steipe, Protein Sci. 8:2245-2250 (1999); Zhu et al., J. Immunol. Methods 231:207-222 (1999); and references cited therein.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or alternatively, under lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:Y, to a polypeptide encoded by a portion of SEQ ID NO:X as defined in columns 8 and 9 of Table 2, and/or to a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z.

The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques. Methods of producing antibodies include, but are not limited to, hybridoma technology, EBV transformation, and other methods discussed herein as well as through the use recombinant DNA technology, as discussed below.

Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells, such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci that in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk−, hgprt− or aprt− cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215 (1993)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Methods commonly known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257 (1983)).

Vectors which use glutamine synthase (GS) or DHFR as the selectable markers can be amplified in the presence of the drugs methionine sulphoximine or methotrexate, respectively. An advantage of glutamine synthase based vectors are the availability of cell lines (e.g., the murine myeloma cell line, NS0) which are glutamine synthase negative. Glutamine synthase expression systems can also function in glutamine synthase expressing cells (e.g. Chinese Hamster Ovary (CHO) cells) by providing additional inhibitor to prevent the functioning of the endogenous gene. A glutamine synthase expression system and components thereof are detailed in PCT publications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; and WO91/06657 which are incorporated in their entireties by reference herein. Additionally, glutamine synthase expression vectors that may be used according to the present invention are commercially available from suppliers, including, for example Lonza Biologics, Inc. (Portsmouth, N.H.). Expression and production of monoclonal antibodies using a GS expression system in murine myeloma cells is described in Bebbington et al., Bio/technology 10:169 (1992) and in Biblia and Robinson Biotechnol. Prog. 11:1 (1995) which are incorporated in their entireties by reference herein.

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by an animal, chemically synthesized, or recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. In addition, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.

The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (said references incorporated by reference in their entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:Y may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides corresponding to SEQ ID NO:Y may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See EP 394,827; and Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. See, for example, Fountoulakis et al., J. Biochem. 270:3958-3964 (1995). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. See, for example, EP A 232,262. Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hIL-5, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, Bennett et al., J. Molecular Recognition 8:52-58 (1995); Johanson et al., J. Biol. Chem. 270:9459-9471 (1995)).

Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the “flag” tag.

The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II (See, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No. WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moiety to antibodies are well known. See, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples. Translation products of the gene of the present invention may be useful as cell-specific markers, or more specifically as cellular markers that are differentially expressed at various stages of differentiation and/or maturation of particular cell types. Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the screening of cellular populations expressing the marker. Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, “panning” with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations of cells, such as might be found with hematological malignancies (i.e. minimal residual disease (MRD) in acute leukemic patients) and “non-self” cells in transplantations to prevent Graft-versus-Host Disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds., (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.16.1.

Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al, eds, (1994), Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, section 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.

Antibodies of the invention may be characterized using immunocytochemisty methods on cells (e.g., mammalian cells, such as CHO cells) transfected with a vector enabling the expression of an antigen or with vector alone using techniques commonly known in the art. Antibodies that bind antigen transfected cells, but not vector-only transfected cells, are antigen specific.

Therapeutic Uses

Table 1D: In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) represented by Table 1A and Table 1D (in the same row as the disease or disorder to be treated is listed in the “Preferred Indications” column of Table 1D) in an amount effective to treat, prevent, or ameliorate the disease or disorder.

As indicated in Table 1D, the polynucleotides, polypeptides, agonists, or antagonists of the present invention (including antibodies) can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides or polypeptides, or agonists or antagonists thereof (including antibodies) could be used to treat the associated disease.

The present invention encompasses methods of preventing, treating, diagnosing, or ameliorating a disease or disorder. In preferred embodiments, the present invention encompasses a method of treating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient in which such treatment, prevention, or amelioration is desired a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) in an amount effective to treat, prevent, diagnose, or ameliorate the disease or disorder. The first and second columns of Table 1D show the “Gene No.” and “cDNA Clone ID No.”, respectively, indicating certain nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof) that may be used in preventing, treating, diagnosing, or ameliorating the disease(s) or disorder(s) indicated in the corresponding row in Column 3 of Table 1D.

In another embodiment, the present invention also encompasses methods of preventing, treating, diagnosing, or ameliorating a disease or disorder listed in the “Preferred Indications” column of Table 1D; comprising administering to a patient combinations of the proteins, nucleic acids, or antibodies of the invention (or fragments or variants thereof), sharing similar indications as shown in the corresponding rows in Column 3 of Table 1D.

The “Preferred Indication” column describes diseases, disorders, and/or conditions that may be treated, prevented, diagnosed, or ameliorated by a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof).

The recitation of “Cancer” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof) may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., leukemias, cancers, and/or as described below under “Hyperproliferative Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D may be used for example, to diagnose, treat, prevent, and/or ameliorate a neoplasm located in a tissue selected from the group consisting of: colon, abdomen, bone, breast, digestive system, liver, pancreas, prostate, peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a pre-neoplastic condition, selected from the group consisting of: hyperplasia (e.g., endometrial hyperplasia and/or as described in the section entitled “Hyperproliferative Disorders”), metaplasia (e.g., connective tissue metaplasia, atypical metaplasia, and/or as described in the section entitled “Hyperproliferative Disorders”), and/or dysplasia (e.g., cervical dysplasia, and bronchopulmonary dysplasia).

In another specific embodiment, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cancer” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a benign dysproliferative disorder selected from the group consisting of: benign tumors, fibrocystic conditions, tissue hypertrophy, and/or as described in the section entitled “Hyperproliferative Disorders”.

The recitation of “Immune/Hematopoietic” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), blood disorders (e.g., as described below under “Immune Activity” “Cardiovascular Disorders” and/or “Blood-Related Disorders”), and infections (e.g., as described below under “Infectious Disease”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having the “Immune/Hematopoietic” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: anemia, pancytopenia, leukopenia, thrombocytopenia, leukemias, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis, granulomatous disease, immune deficiency, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, systemic lupus erythematosis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis, Lyme Disease, and allergies.

The recitation of “Reproductive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the reproductive system (e.g., as described below under “Reproductive System Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Reproductive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cryptorchism, prostatitis, inguinal hernia, varicocele, leydig cell tumors, verrucous carcinoma, prostatitis, malacoplakia, Peyronie's disease, penile carcinoma, squamous cell hyperplasia, dysmenorrhea, ovarian adenocarcinoma, Turner's syndrome, mucopurulent cervicitis, Sertoli-leydig tumors, ovarian cancer, uterine cancer, pelvic inflammatory disease, testicular cancer, prostate cancer, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, testicular atrophy, testicular feminization, anorchia, ectopic testis, epididymitis, orchitis, gonorrhea, syphilis, testicular torsion, vasitis nodosa, germ cell tumors, stromal tumors, dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding, cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, cervical neoplasms, pseudohermaphroditism, and premenstrual syndrome.

The recitation of “Musculoskeletal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the immune system (e.g., as described below under “Immune Activity”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Musculoskeletal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bone cancers (e.g., osteochondromas, benign chondromas, chondroblastoma, chondromyxoid fibromas, osteoid osteomas, giant cell tumors, multiple myeloma, osteosarcomas), Paget's Disease, rheumatoid arthritis, systemic lupus erythematosus, osteomyelitis, Lyme Disease, gout, bursitis, tendonitis, osteoporosis, osteoarthritis, muscular dystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis.

The recitation of “Cardiovascular” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), and disorders of the cardiovascular system (e.g., as described below under “Cardiovascular Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Cardiovascular” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: myxomas, fibromas, rhabdomyomas, cardiovascular abnormalities (e.g., congenital heart defects, cerebral arteriovenous malformations, septal defects), heart disease (e.g., heart failure, congestive heart disease, arrhythmia, tachycardia, fibrillation, pericardial Disease, endocarditis), cardiac arrest, heart valve disease (e.g., stenosis, regurgitation, prolapse), vascular disease (e.g., hypertension, coronary artery disease, angina, aneurysm, arteriosclerosis, peripheral vascular disease), hyponatremia, hypernatremia, hypokalemia, and hyperkalemia.

The recitation of “Mixed Fetal” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Mixed Fetal” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: spina bifida, hydranencephaly, neurofibromatosis, fetal alcohol syndrome, diabetes mellitus, PKU, Down's syndrome, Patau syndrome, Edwards syndrome, Turner syndrome, Apert syndrome, Carpenter syndrome, Conradi syndrome, Crouzon syndrome, cutis laxa, Cornelia de Lange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome, Kartagener syndrome, Meckel-Gruber syndrome, Noonan syndrome, Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome, Smith-Lemli-Opitz syndrome, thromocytopenia-absent radius (TAR) syndrome, Treacher Collins syndrome, Williams syndrome, Hirschsprung's disease, Meckel's diverticulum, polycystic kidney disease, Turner's syndrome, and gonadal dysgenesis, Klippel-Feil syndrome, Ostogenesis imperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumor, neuroblastoma, and retinoblastoma.

The recitation of “Excretory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and renal disorders (e.g., as described below under “Renal Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Excretory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: bladder cancer, prostate cancer, benign prostatic hyperplasia, bladder disorders (e.g., urinary incontinence, urinary retention, urinary obstruction, urinary tract Infections, interstitial cystitis, prostatitis, neurogenic bladder, hematuria), renal disorders (e.g., hydronephrosis, proteinuria, renal failure, pyelonephritis, urolithiasis, reflux nephropathy, and unilateral obstructive uropathy).

The recitation of “Neural/Sensory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the nervous system (e.g., as described below under “Neural Activity and Neurological Diseases”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Neural/Sensory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: brain cancer (e.g., brain stem glioma, brain tumors, central nervous system (Primary) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, and cerebral astrocytoma, neurodegenerative disorders (e.g., Alzheimer's Disease, Creutzfeldt-Jakob Disease, Parkinson's Disease, and Idiopathic Presenile Dementia), encephalomyelitis, cerebral malaria, meningitis, metabolic brain diseases (e.g., phenylketonuria and pyruvate carboxylase deficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS Dementia Complex, schizophrenia, attention deficit disorder, hyperactive attention deficit disorder, autism, and obsessive compulsive disorders.

The recitation of “Respiratory” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Respiratory” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of the respiratory system such as larynx cancer, pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamous cell carcinomas, small cell (oat cell) carcinomas, large cell carcinomas, and adenocarcinomas. Allergic reactions, cystic fibrosis, sarcoidosis, histiocytosis X, infiltrative lung diseases (e.g., pulmonary fibrosis and lymphoid interstitial pneumonia), obstructive airway diseases (e.g., asthma, emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis and asbestosis), pneumonia, and pleurisy.

The recitation of “Endocrine” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the respiratory system (e.g., as described below under “Respiratory Disorders”), renal disorders (e.g., as described below under “Renal Disorders”), and disorders of the endocrine system (e.g., as described below under “Endocrine Disorders”.

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having an “Endocrine” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: cancers of endocrine tissues and organs (e.g., cancers of the hypothalamus, pituitary gland, thyroid gland, parathyroid glands, pancreas, adrenal glands, ovaries, and testes), diabetes (e.g., diabetes insipidus, type I and type II diabetes mellitus), obesity, disorders related to pituitary glands (e.g., hyperpituitarism, hypopituitarism, and pituitary dwarfism), hypothyroidism, hyperthyroidism, goiter, reproductive disorders (e.g. male and female infertility), disorders related to adrenal glands (e.g., Addison's Disease, corticosteroid deficiency, and Cushing's Syndrome), kidney cancer (e.g., hypernephroma, transitional cell cancer, and Wilm's tumor), diabetic nephropathy, interstitial nephritis, polycystic kidney disease, glomerulonephritis (e.g., IgM mesangial proliferative glomerulonephritis and glomerulonephritis caused by autoimmune disorders; such as Goodpasture's syndrome), and nephrocalcinosis.

The recitation of “Digestive” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”) and diseases or disorders of the gastrointestinal system (e.g., as described below under “Gastrointestinal Disorders”.

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Digestive” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepatic encephalopathy, portal hypertension, cholelithiasis, cancer of the digestive system (e.g., biliary tract cancer, stomach cancer, colon cancer, gastric cancer, pancreatic cancer, cancer of the bile duct, tumors of the colon (e.g., polyps or cancers), and cirrhosis), pancreatitis, ulcerative disease, pyloric stenosis, gastroenteritis, gastritis, gastric atropy, benign tumors of the duodenum, distension, irritable bowel syndrome, malabsorption, congenital disorders of the small intestine, bacterial and parasitic infection, megacolon, Hirschsprung's disease, aganglionic megacolon, acquired megacolon, colitis, anorectal disorders (e.g., anal fistulas, hemorrhoids), congenital disorders of the liver (e.g., Wilson's disease, hemochromatosis, cystic fibrosis, biliary atresia, and alpha1-antitrypsin deficiency), portal hypertension, cholelithiasis, and jaundice.

The recitation of “Connective/Epithelial” in the “Preferred Indication” column indicates that the corresponding nucleic acid and protein, or antibody against the same, of the invention (or fragment or variant thereof), may be used for example, to diagnose, treat, prevent, and/or ameliorate diseases and/or disorders relating to neoplastic diseases (e.g., as described below under “Hyperproliferative Disorders”), cellular and genetic abnormalities (e.g., as described below under “Diseases at the Cellular Level”), angiogenesis (e.g., as described below under “Anti-Angiogenesis Activity”), and or to promote or inhibit regeneration (e.g., as described below under “Regeneration”), and wound healing (e.g., as described below under “Wound Healing and Epithelial Cell Proliferation”).

In specific embodiments, a protein, nucleic acid, or antibody of the invention (or fragment or variant thereof) having a “Connective/Epithelial” recitation in the “Preferred Indication” column of Table 1D, may be used for example, to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: connective tissue metaplasia, mixed connective tissue disease, focal epithelial hyperplasia, epithelial metaplasia, mucoepithelial dysplasia, graft v. host disease, polymyositis, cystic hyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer's disease, lymphoproliferative disorder, Waldenstron's macroglobulinemia, Crohn's disease, pernicious anemia, idiopathic Addison's disease, glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetes mellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease, wound healing, relapsing polychondritis, vasculitis, polyarteritis nodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderma, CREST syndrome, Sjogren's syndrome, polymyositis, dermatomyositis, mixed connective tissue disease, relapsing polychondritis, vasculitis, Henoch-Schonlein syndrome, erythema nodosum, polyarteritis nodosa, temporal (giant cell) arteritis, Takayasu's arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, cellulitis, keloids, Ehler Danlos syndrome, Marfan syndrome, pseudoxantoma elasticum, osteogenese imperfecta, chondrodysplasias, epidermolysis bullosa, Alport syndrome, and cutis laxa.

Table 1E also provides information regarding biological activities and preferred therapeutic uses (i.e. see, “Preferred Indications” column) for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Table 1E also provides information regarding assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column (“Gene No.”) provides the gene number in the application for each clone identifier. The second column (“cDNA ATCC™ Deposit No:Z”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B.1, 1B.2, 1C, and 1D. The third column (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A and 1B.1). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides). The fifth column (“Exemplary Activity Assay”) further describes the corresponding biological activity and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The sixth column (“Preferred Indications”) describes particular embodiments of the invention as well as indications (e.g. pathologies, diseases, disorders, abnormalities, etc.) for which polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) may be used in detecting, diagnosing, preventing, and/or treating.

Tables 1E.1 and 1E.2 also provide information regarding biological activities and preferred therapeutic uses (i.e. see, “Preferred Indications” column) for polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof). Tables 1E.2 also provide information regarding assays which may be used to test polynucleotides and polypeptides of the invention (including antibodies, agonists, and/or antagonists thereof) for the corresponding biological activities. The first column of Table 1E.1 (“Gene No.”) provides the gene number in the application for each clone identifier. The second column of Table 1E.1 (“cDNA Clone ID”) provides the unique clone identifier for each clone as previously described and indicated in Tables 1A, 1B.1, 1B.2, and 1C. The third column of Table 1E.1 (“AA SEQ ID NO:Y”) indicates the Sequence Listing SEQ ID Number for polypeptide sequences encoded by the corresponding cDNA clones (also as indicated in Tables 1A, 1B.1, 1B.2, and 2). The fourth column (“Biological Activity”) indicates a biological activity corresponding to the indicated polypeptides (or polynucleotides encoding said polypeptides).

In Table 1E.2, each of the biological activities of Table 1E.1 are listed by “Biological Activity Number” and the corresponding “Biological Activity” and are followed by an “Exemplary Activity Assay” column and a “Preferred Indication” column; however, for some biological activities no “Exemplary Activity Assay” or “Preferred Indication” is given. The “Exemplary Activity Assay” column describes the biological activity listed in the column that precedes it and also provides information pertaining to the various types of assays which may be performed to test, demonstrate, or quantify the corresponding biological activity. The “Preferred Indication” column also refers to the biological activity listed in the preceding column and describes disease(s) or disorder(s) that may be detected, diagnosed, prevented, treated, or ameliorated by the nucleic acids and proteins (or antibodies against the same) of the invention (including polynucleotide, polypeptide, and antibody fragments or variants thereof).

The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to detect, prevent, diagnose, prognosticate, treat, inhibit and/or ameliorate diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or momore of the diseases, disorders, or conditions described herein, such as immune, cardiovascular, cancer, and other proliferative diseases and disorders. The treatment and/or prevention of diseases, disorders, or conditions associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions, such as immune, cardiovascular, cancer, and other proliferative diseases and disorders. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

In a specific and preferred embodiment, the present invention is directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more diseases, disorders, or conditions, including but not limited to: neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions., and/or as described elsewhere herein. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (e.g., antibodies directed to the full length protein expressed on the cell surface of a mammalian cell; antibodies directed to an epitope of a polypeptide of the invention (such as, for example, a predicted linear epitope shown in Table 1B.1 (such as an epitope shown in column 7 of Table 1B.1); or a conformational epitope, including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to detect, diagnose, inhibit, prevent, treat, prognosticate, and/or ameliorate diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein (such as immune, cardiovascular, cancer, and other proliferative diseases and disorders). The treatment and/or prevention of diseases, disorders, or conditions (such as immune, cardiovascular, cancer, and other proliferative diseases and disorders) associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of diseases and disorders (such as immune, cardiovascular, cancer, and other proliferative diseases and disorders) related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides of the invention, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder (such an immune, cardiovascular, cancer, and other proliferative disease or disorder) associated with aberrant expression and/or activity of a polypeptide of the invention, by way of gene therapy. Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acids produce their encoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.

For general reviews of the methods of gene therapy, see Goldspiel et al., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred embodiment, the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host. In particular, such nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue-specific. In another particular embodiment, nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). In specific embodiments, the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.

Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid-carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; BIOLISTIC™, DUPONT™), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc. In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

In a specific embodiment, viral vectors which contain nucleic acid sequences encoding an antibody of the invention are used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient. More detail about retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993); Cline, Pharmac. Ther. 29:69-92 m (1985) and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not disrupted. The technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.

In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In an embodiment in which recombinant cells are used in gene therapy, nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO 94/08598; Stemple and Anderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229 (1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by the presence or absence of an appropriate inducer of transcription.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably a polypeptide or antibody of the invention. In a preferred embodiment, the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.

Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.

Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.

In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, e.g., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; BIOLISTIC™, DUPONT™), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox like peptide which is known to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

The amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, prognosticate, or monitor diseases, disorders, and/or conditions (such as immune, cardiovascular, cancer, and other proliferative diseases, disorders, and conditions) associated with the aberrant expression and/or activity of a polypeptide of the invention. The invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disease or disorder (such an immune, cardiovascular, cancer, or other proliferative disease or disorders), comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disease or disorder. With respect to cancer and other hyperproliferative diseases and disorders (such as immunogenic cancer or cancer of the cardiovascular system), the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer or other hyperproliferative disease (such as immunogenic cancer or cancer of the cardiovascular system).

Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

One facet of the invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide of interest in an animal, preferably a mammal and most preferably a human. In one embodiment, diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99 mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

Depending on several variables, including the type of label used and the mode of administration, the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.

In a specific embodiment, the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al., U.S. Pat. No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).

Kits

The present invention provides kits that can be used in the above methods. In one embodiment, a kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In a specific embodiment, the kits of the present invention contain a substantially isolated polypeptide comprising an epitope that is specifically immunoreactive with an antibody included in the kit. Preferably, the kits of the present invention further comprise a control antibody that does not react with the polypeptide of interest. In another specific embodiment, the kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).

In another specific embodiment of the present invention, the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may include a substantially isolated polypeptide antigen comprising an epitope that is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry). In specific embodiments, the kit may include a recombinantly produced or chemically synthesized polypeptide antigen. The polypeptide antigen of the kit may also be attached to a solid support.

In a more specific embodiment the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit for use in screening serum containing antigens of the polypeptide of the invention. The diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In a specific embodiment, the antibody may be a monoclonal antibody. The detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention. After binding with specific antigen antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (SIGMA™, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying out this diagnostic method. The kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerous ways as reagents. The following description should be considered exemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents, based on actual sequence data (repeat polymorphisms), are presently available. Each sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome, thus each polynucleotide of the present invention can routinely be used as a chromosome marker using techniques known in the art. Table 1B.1, column 8 provides the chromosome location of some of the polynucleotides of the invention.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably at least 15 bp (e.g., 15-25 bp) from the sequences shown in SEQ ID NO:X. Primers can optionally be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to SEQ ID NO:X will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments. Other gene mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, preselection by hybridization to construct chromosome specific-cDNA libraries, and computer mapping techniques (See, e.g., Shuler, Trends Biotechnol 16:456-459 (1998) which is hereby incorporated by reference in its entirety).

Precise chromosomal location of the polynucleotides can also be achieved using fluorescence in situ hybridization (FISH) of a metaphase chromosomal spread. This technique uses polynucleotides as short as 500 or 600 bases; however, polynucleotides 2,000-4,000 by are preferred. For a review of this technique, see Verma et al., “Human Chromosomes: a Manual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes). Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross hybridization during chromosomal mapping.

Thus, the present invention also provides a method for chromosomal localization which involves (a) preparing PCR primers from the polynucleotide sequences in Table 1B.1 and/or 2 and SEQ ID NO:X and (b) screening somatic cell hybrids containing individual chromosomes.

The polynucleotides of the present invention would likewise be useful for radiation hybrid mapping, HAPPY mapping, and long range restriction mapping. For a review of these techniques and others known in the art, see, e.g. Dear, “Genome Mapping: A Practical Approach,” IRL Press at Oxford University Press, London (1997); Aydin, J. Mol. Med. 77:691-694 (1999); Hacia et al., Mol. Psychiatry. 3:483-492 (1998); Herrick et al., Chromosome Res. 7:409-423 (1999); Hamilton et al., Methods Cell Biol. 62:265-280 (2000); and/or Ott, J. Hered. 90:68-70 (1999) each of which is hereby incorporated by reference in its entirety.

Once a polynucleotide has been mapped to a precise chromosomal location, the physical position of the polynucleotide can be used in linkage analysis. Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease. (Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library)). Table 1B.1 (such as column 9 of Table 1B.1) provides an OMIM reference identification number of diseases associated with the cytologic band disclosed in Table 1B.1 (such as column 8 of Table 1B.1), as determined using techniques described herein and by reference to Table 5. Assuming 1 megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.

Thus, once coinheritance is established, differences in a polynucleotide of the invention and the corresponding gene between affected and unaffected individuals can be examined. First, visible structural alterations in the chromosomes, such as deletions or translocations, are examined in chromosome spreads or by PCR. If no structural alterations exist, the presence of point mutations are ascertained. Mutations observed in some or all affected individuals, but not in normal individuals, indicates that the mutation may cause the disease. However, complete sequencing of the polypeptide and the corresponding gene from several normal individuals is required to distinguish the mutation from a polymorphism. If a new polymorphism is identified, this polymorphic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affected individuals as compared to unaffected individuals can be assessed using the polynucleotides of the invention. Any of these alterations (altered expression, chromosomal rearrangement, or mutation) can be used as a diagnostic or prognostic marker. Diagnostic and prognostic methods, kits and reagents encompassed by the present invention are briefly described below and more thoroughly elsewhere herein (see e.g., the sections labeled “Antibodies”, “Diagnostic Assays”, and “Methods for Detecting Diseases”).

Thus, the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder. Additional non-limiting examples of diagnostic methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., Example 12).

In still another embodiment, the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject. In a general embodiment, the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide of the invention and a suitable container. In a specific embodiment, the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the invention, where each probe has one strand containing a 31′mer-end internal to the region. In a further embodiment, the probes may be useful as primers for polymerase chain reaction amplification.

Where a diagnosis of a related disorder, including, for example, diagnosis of a tumor, has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide of the invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.

By “measuring the expression level of polynucleotides of the invention” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the invention or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the related disorder or being determined by averaging levels from a population of individuals not having a related disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source that contains polypeptide of the present invention or the corresponding mRNA. As indicated, biological samples include body fluids (such as semen, lymph, vaginal pool, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

The method(s) provided above may preferably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides of the invention are attached to a solid support. In one exemplary method, the support may be a “gene chip” or a “biological chip” as described in U.S. Pat. Nos. 5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip with polynucleotides of the invention attached may be used to identify polymorphisms between the isolated polynucleotide sequences of the invention, with polynucleotides isolated from a test subject. The knowledge of such polymorphisms (i.e. their location, as well as, their existence) would be beneficial in identifying disease loci for many disorders, such as for example, in neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, digestive disorders, metabolic disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. Such a method is described in U.S. Pat. Nos. 5,858,659 and 5,856,104. The US patents referenced supra are hereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the present invention that are chemically synthesized, or reproduced as peptide nucleic acids (PNA), or according to other methods known in the art. The use of PNAs would serve as the preferred form if the polynucleotides of the invention are incorporated onto a solid support, or gene chip. For the purposes of the present invention, a peptide nucleic acid (PNA) is a polyamide type of DNA analog and the monomeric units for adenine, guanine, thymine and cytosine are available commercially (Perceptive Biosystems). Certain components of DNA, such as phosphorus, phosphorus oxides, or deoxyribose derivatives, are not present in PNAs. As disclosed by Nielsen et al., Science 254, 1497 (1991); and Egholm et al., Nature 365, 666 (1993), PNAs bind specifically and tightly to complementary DNA strands and are not degraded by nucleases. In fact, PNA binds more strongly to DNA than DNA itself does. This is probably because there is no electrostatic repulsion between the two strands, and also the polyamide backbone is more flexible. Because of this, PNA/DNA duplexes bind under a wider range of stringency conditions than DNA/DNA duplexes, making it easier to perform multiplex hybridization. Smaller probes can be used than with DNA due to the strong binding. In addition, it is more likely that single base mismatches can be determined with PNA/DNA hybridization because a single mismatch in a PNA/DNA 15-mer lowers the melting point (T_(m)) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, the absence of charge groups in PNA means that hybridization can be done at low ionic strengths and reduce possible interference by salt during the analysis.

The compounds of the present invention have uses which include, but are not limited to, detecting cancer in mammals. In particular the invention is useful during diagnosis of pathological cell proliferative neoplasias which include, but are not limited to: acute myelogenous leukemias including acute monocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute erythroleukemia, acute megakaryocytic leukemia, and acute undifferentiated leukemia, etc.; and chronic myelogenous leukemias including chronic myelomonocytic leukemia, chronic granulocytic leukemia, etc. Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans. Particularly preferred are humans.

Pathological cell proliferative disorders are often associated with inappropriate activation of proto-oncogenes. (Gelmann, E. P. et al., “The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology,” in Neoplastic Diseases of the Blood, Vol 1., Wiernik, P. H. et al. eds., 161-182 (1985)). Neoplasias are now believed to result from the qualitative alteration of a normal cellular gene product, or from the quantitative modification of gene expression by insertion into the chromosome of a viral sequence, by chromosomal translocation of a gene to a more actively transcribed region, or by some other mechanism. (Gelmann et al., supra) It is likely that mutated or altered expression of specific genes is involved in the pathogenesis of some leukemias, among other tissues and cell types. (Gelmann et al., supra) Indeed, the human counterparts of the oncogenes involved in some animal neoplasias have been amplified or translocated in some cases of human leukemia and carcinoma. (Gelmann et al., supra)

For example, c-myc expression is highly amplified in the non-lymphocytic leukemia cell line HL-60. When HL-60 cells are chemically induced to stop proliferation, the level of c-myc is found to be downregulated. (International Publication Number WO 91/15580). However, it has been shown that exposure of HL-60 cells to a DNA construct that is complementary to the 5′ end of c-myc or c-myb blocks translation of the corresponding mRNAs which downregulates expression of the c-myc or c-myb proteins and causes arrest of cell proliferation and differentiation of the treated cells. (International Publication Number WO 91/15580; Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al., Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisan would appreciate the present invention's usefulness is not be limited to treatment, prevention, and/or prognosis of proliferative disorders of cells and tissues of hematopoietic origin, in light of the numerous cells and cell types of varying origins which are known to exhibit proliferative phenotypes.

In addition to the foregoing, a polynucleotide of the present invention can be used to control gene expression through triple helix formation or through antisense DNA or RNA. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); and Dervan et al., Science 251: 1360 (1991). Both methods rely on binding of the polynucleotide to a complementary DNA or RNA. For these techniques, preferred polynucleotides are usually oligonucleotides 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. The oligonucleotide described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of polypeptide of the present invention antigens. Both techniques are effective in model systems, and the information disclosed herein can be used to design antisense or triple helix polynucleotides in an effort to treat disease, and in particular, for the treatment of proliferative diseases and/or conditions. Non-limiting antisense and triple helix methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the section labeled “Antisense and Ribozyme (Antagonists)”).

Polynucleotides of the present invention are also useful in gene therapy. One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect. The polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner. Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell. Additional non-limiting examples of gene therapy methods encompassed by the present invention are more thoroughly described elsewhere herein (see, e.g., the sections labeled “Gene Therapy Methods”, and Examples 16, 17 and 18).

The polynucleotides are also useful for identifying individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The polynucleotides of the present invention can be used as additional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.

Forensic biology also benefits from using DNA-based identification techniques as disclosed herein. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk, lymph, pulmonary sputum or surfactant, urine, fecal matter, etc., can be amplified using PCR. In one prior art technique, gene sequences amplified from polymorphic loci, such as DQa class II HLA gene, are used in forensic biology to identify individuals. (Erlich, H., PCR Technology, Freeman and Co. (1992)). Once these specific polymorphic loci are amplified, they are digested with one or more restriction enzymes, yielding an identifying set of bands on a Southern blot probed with DNA corresponding to the DQa class II HLA gene. Similarly, polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.

There is also a need for reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin. Appropriate reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention, specific to tissues, including but not limited to those shown in Table 1B.2. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination. Additional non-limiting examples of such uses are further described herein.

The polynucleotides of the present invention are also useful as hybridization probes for differential identification of the tissue(s) or cell type(s) present in a biological sample. Similarly, polypeptides and antibodies directed to polypeptides of the present invention are useful to provide immunological probes for differential identification of the tissue(s) (e.g., immunohistochemistry assays) or cell type(s) (e.g., immunocytochemistry assays). In addition, for a number of disorders of the above tissues or cells, significantly higher or lower levels of gene expression of the polynucleotides/polypeptides of the present invention may be detected in certain tissues (e.g., tissues expressing polypeptides and/or polynucleotides of the present invention, for example, those disclosed in Table 1B.2 (such as in column 5 of Table 1B.2), and/or cancerous and/or wounded tissues) or bodily fluids (e.g., semen, lymph, vaginal pool, serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a “standard” gene expression level, i.e., the expression level in healthy tissue from an individual not having the disorder.

Thus, the invention provides a diagnostic method of a disorder, which involves: (a) assaying gene expression level in cells or body fluid of an individual; (b) comparing the gene expression level with a standard gene expression level, whereby an increase or decrease in the assayed gene expression level compared to the standard expression level is indicative of a disorder.

In the very least, the polynucleotides of the present invention can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to “subtract-out” known sequences in the process of discovering novel polynucleotides, for selecting and making oligomers for attachment to a “gene chip” or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.

Polypeptides and antibodies directed to polypeptides of the invention are useful to provide immunological probes for differential identification of the tissue(s) (e.g., immunohistochemistry assays such as, for example, ABC immunoperoxidase (Hsu et al., J. Histochem. Cytochem. 29:577-580 (1981)) or cell type(s) (e.g., immunocytochemistry assays).

Antibodies can be used to assay levels of polypeptides encoded by polynucleotides of the invention in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase; radioisotopes, such as iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I) (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying levels of polypeptide of the present invention in a biological sample, proteins can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc, (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In, ¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti) gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for immune system disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of ⁹⁹ mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which express the polypeptide encoded by a polynucleotide of the invention. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (e.g., polypeptides encoded by polynucleotides of the invention and/or antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention in association with toxins or cytotoxic prodrugs.

By “toxin” is meant one or more compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. “Toxin” also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi, or other radioisotopes such as for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, ⁹⁰Yttrium, ¹¹⁷Tin, ¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. In a specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope ⁹⁰Y. In another specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope ¹¹¹In. In a further specific embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention or antibodies of the invention in association with the radioisotope ¹³¹I.

Techniques known in the art may be applied to label polypeptides of the invention (including antibodies). Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety).

Thus, the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression level of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the assayed polypeptide expression level with a standard polypeptide expression level, whereby an increase or decrease in the assayed polypeptide expression level compared to the standard expression level is indicative of a disorder. With respect to cancer, the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the cancer.

Moreover, polypeptides of the present invention can be used to treat or prevent diseases or conditions such as, for example, neural disorders, immune system disorders, muscular disorders, reproductive disorders, gastrointestinal disorders, pulmonary disorders, cardiovascular disorders, renal disorders, proliferative disorders, and/or cancerous diseases and conditions. For example, patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin), to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repair proteins), to inhibit the activity of a polypeptide (e.g., an oncogene or tumor supressor), to activate the activity of a polypeptide (e.g., by binding to a receptor), to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation), or to bring about a desired response (e.g., blood vessel growth inhibition, enhancement of the immune response to proliferative cells or tissues).

Similarly, antibodies directed to a polypeptide of the present invention can also be used to treat disease (as described supra, and elsewhere herein). For example, administration of an antibody directed to a polypeptide of the present invention can bind, and/or neutralize the polypeptide, and/or reduce overproduction of the polypeptide. Similarly, administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art. Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell. Moreover, the polypeptides of the present invention can be used to test the biological activities described herein.

Diagnostic Assays

The compounds of the present invention are useful for diagnosis, treatment, prevention and/or prognosis of various disorders in mammals, preferably humans. Such disorders include, but are not limited to, those described in the legends for Tables 1D, 1E.1, and 1F, and as indicated in the “Preferred Indications” columns in Table 1D, 1E.1, and 1F; and, also as described herein under the section heading “Biological Activities.” Such disorders also include, but are not limited to, those related to biological activities described in Tables 1E, 1E.1, and 1E.2.

For a number of disorders, substantially altered (increased or decreased) levels of gene expression can be detected in tissues, cells or bodily fluids (e.g., sera, plasma, urine, semen, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a “standard” gene expression level, that is, the expression level in tissues or bodily fluids from an individual not having the disorder. Thus, the invention provides a diagnostic method useful during diagnosis of a disorder, which involves measuring the expression level of the gene encoding the polypeptide in tissues, cells or body fluid from an individual and comparing the measured gene expression level with a standard gene expression level, whereby an increase or decrease in the gene expression level(s) compared to the standard is indicative of a disorder. These diagnostic assays may be performed in vivo or in vitro, such as, for example, on blood samples, biopsy tissue or autopsy tissue.

The present invention is also useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed gene expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level.

In certain embodiments, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognosticate diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2 (such as column 5) (Tissue Distribution Library Code).

By “assaying the expression level of the gene encoding the polypeptide” is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the invention or the level of the mRNA encoding the polypeptide of the invention in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the polypeptide expression level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having the disorder. As will be appreciated in the art, once a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained from an individual, cell line, tissue culture, or other source containing polypeptides of the invention (including portions thereof) or mRNA. As indicated, biological samples include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) and tissue sources found to express the full length or fragments thereof of a polypeptide or mRNA. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

Total cellular RNA can be isolated from a biological sample using any suitable technique such as the single-step guanidinium-thiocyanate-phenol-chloroform method described in Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels of mRNA encoding the polypeptides of the invention are then assayed using any appropriate method. These include Northern blot analysis, S1 nuclease mapping, the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR), and reverse transcription in combination with the ligase chain reaction (RT-LCR).

The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of polypeptides of the invention, in a biological sample (e.g., cells and tissues), including determination of normal and abnormal levels of polypeptides. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of polypeptides of the invention compared to normal control tissue samples may be used to detect the presence of tumors. Assay techniques that can be used to determine levels of a polypeptide, such as a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Assaying polypeptide levels in a biological sample can occur using any art-known method.

Assaying polypeptide levels in a biological sample can occur using antibody-based techniques. For example, polypeptide expression in tissues can be studied with classical immunohistological methods (Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.

The tissue or cell type to be analyzed will generally include those which are known, or suspected, to express the gene of inteest (such as, for example, cancer). The protein isolation methods employed herein may, for example, be such as those described in Harlow and Lane (Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety. The isolated cells can be derived from cell culture or from a patient. The analysis of cells taken from culture may be a necessary step in the assessment of cells that could be used as part of a cell-based gene therapy technique or, alternatively, to test the effect of compounds on the expression of the gene.

For example, antibodies, or fragments of antibodies, such as those described herein, may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.

In a preferred embodiment, antibodies, or fragments of antibodies directed to any one or all of the predicted epitope domains of the polypeptides of the invention (shown in Table 1B.1 such as column 7 of Table 1B.1) may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.

In an additional preferred embodiment, antibodies, or fragments of antibodies directed to a conformational epitope of a polypeptide of the invention may be used to quantitatively or qualitatively detect the presence of gene products or conserved variants or peptide fragments thereof. This can be accomplished, for example, by immunofluorescence techniques employing a fluorescently labeled antibody coupled with light microscopic, flow cytometric, or fluorimetric detection.

The antibodies (or fragments thereof), and/or polypeptides of the present invention may, additionally, be employed histologically, as in immunofluorescence, immunoelectron microscopy or non-immunological assays, for in situ detection of gene products or conserved variants or peptide fragments thereof. In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody or polypeptide of the present invention. The antibody (or fragment thereof) or polypeptide is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of the gene product, or conserved variants or peptide fragments, or polypeptide binding, but also its distribution in the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Immunoassays and non-immunoassays for gene products or conserved variants or peptide fragments thereof will typically comprise incubating a sample, such as a biological fluid, a tissue extract, freshly harvested cells, or lysates of cells which have been incubated in cell culture, in the presence of a detectably labeled antibody capable of binding gene products or conserved variants or peptide fragments thereof, and detecting the bound antibody by any of a number of techniques well-known in the art.

The biological sample may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, or other solid support that is capable of immobilizing cells, cell particles or soluble proteins. The support may then be washed with suitable buffers followed by treatment with the detectably labeled antibody or detectable polypeptide of the invention. The solid phase support may then be washed with the buffer a second time to remove unbound antibody or polypeptide. Optionally the antibody is subsequently labeled. The amount of bound label on solid support may then be detected by conventional means.

By “solid phase support or carrier” is intended any support capable of binding an antigen or an antibody. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of antibody or antigen polypeptide may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

In addition to assaying polypeptide levels or polynucleotide levels in a biological sample obtained from an individual, polypeptide or polynucleotide can also be detected in vivo by imaging. For example, in one embodiment of the invention, polypeptides and/or antibodies of the invention are used to image diseased cells, such as neoplasms. In another embodiment, polynucleotides of the invention (e.g., polynucleotides complementary to all or a portion of an mRNA) and/or antibodies (e.g., antibodies directed to any one or a combination of the epitopes of a polypeptide of the invention, antibodies directed to a conformational epitope of a polypeptide of the invention, or antibodies directed to the full length polypeptide expressed on the cell surface of a mammalian cell) are used to image diseased or neoplastic cells.

Antibody labels or markers for in vivo imaging of polypeptides of the invention include those detectable by X-radiography, NMR, MRI, CAT-scans or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma. Where in vivo imaging is used to detect enhanced levels of polypeptides for diagnosis in humans, it may be preferable to use human antibodies or “humanized” chimeric monoclonal antibodies. Such antibodies can be produced using techniques described herein or otherwise known in the art. For example methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985).

Additionally, any polypeptides of the invention whose presence can be detected, can be administered. For example, polypeptides of the invention labeled with a radio-opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further, such polypeptides can be utilized for in vitro diagnostic procedures.

A polypeptide-specific antibody or antibody fragment that has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for a disorder. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of ^(99m)Tc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the antigenic protein. In vivo tumor imaging is described in S. W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

With respect to antibodies, one of the ways in which an antibody of the present invention can be detectably labeled is by linking the same to a reporter enzyme and using the linked product in an enzyme immunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, Md.); Voller et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth. Enzymol. 73:482-523 (1981); Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The reporter enzyme which is bound to the antibody will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means. Reporter enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Additionally, the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the reporter enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of other immunoassays. For example, by radioactively labeling the antibodies or antibody fragments, it is possible to detect polypeptides through the use of a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by means including, but not limited to, a gamma counter, a scintillation counter, or autoradiography.

It is also possible to label the antibody with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence. Among the most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

The antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.

Methods for Detecting Diseases

In general, a disease may be detected in a patient based on the presence of one or more proteins of the invention and/or polynucleotides encoding such proteins in a biological sample (for example, blood, sera, urine, and/or tumor biopsies) obtained from the patient. In other words, such proteins may be used as markers to indicate the presence or absence of a disease or disorder, including cancer and/or as described elsewhere herein. In addition, such proteins may be useful for the detection of other diseases and cancers. The binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample. Polynucleotide primers and probes may be used to detect the level of mRNA encoding polypeptides of the invention, which is also indicative of the presence or absence of a disease or disorder, including cancer. In general, polypeptides of the invention should be present at a level that is at least three fold higher in diseased tissue than in normal tissue.

There are a variety of assay formats known to those of ordinary skill in the art for using a binding agent to detect polypeptide markers in a sample. See, e.g., Harlow and Lane, supra. In general, the presence or absence of a disease in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.

In a preferred embodiment, the assay involves the use of a binding agent(s) immobilized on a solid support to bind to and remove the polypeptide of the invention from the remainder of the sample. The bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex. Such detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin. Alternatively, a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample. The extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent. Suitable polypeptides for use within such assays include polypeptides of the invention and portions thereof, or antibodies, to which the binding agent binds, as described above.

The solid support may be any material known to those of skill in the art to which polypeptides of the invention may be attached. For example, the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane. Alternatively, the support may be a bead or disc, such as glass fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride. The support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Pat. No. 5,359,681. The binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature. In the context of the present invention, the term “immobilization” refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the agent and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is preferred. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for the suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day. In general, contacting a well of plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 μg, and preferably about 100 ng to about 1 μg, is sufficient to immobilize an adequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent. For example, the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).

Gene Therapy Methods

Also encompassed by the invention are gene therapy methods for treating or preventing disorders, diseases and conditions. The gene therapy methods relate to the introduction of nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into an animal to achieve expression of the polypeptide of the present invention. This method requires a polynucleotide which codes for a polypeptide of the present invention operatively linked to a promoter and any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are known in the art, see, for example, WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) comprising a promoter operably linked to a polynucleotide of the present invention ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide of the present invention. Such methods are well-known in the art. For example, see Belldegrun, A., et al., J. Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., Cancer Research 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153: 4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995); Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al., Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer Gene Therapy 3: 31-38 (1996)), which are herein incorporated by reference. In one embodiment, the cells which are engineered are arterial cells. The arterial cells may be reintroduced into the patient through direct injection to the artery, the tissues surrounding the artery, or through catheter injection.

As discussed in more detail below, the polynucleotide constructs can be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, and the like). The polynucleotide constructs may be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

In one embodiment, the polynucleotide of the present invention is delivered as a naked polynucleotide. The term “naked” polynucleotide, DNA or RNA refers to sequences that are free from any delivery vehicle that acts to assist, promote or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, LIPOFECTIN™ or precipitating agents and the like. However, the polynucleotide of the present invention can also be delivered in liposome formulations and LIPOFECTIN™ formulations and the like can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.

The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from STRATAGENE™; pSVK3, pBPV, pMSG and pSVL available from PHARMACIA™; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available from Invitrogen. Other suitable vectors will be readily apparent to the skilled artisan.

Any strong promoter known to those skilled in the art can be used for driving the expression of the polynucleotide sequence. Suitable promoters include adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs; the b-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter for the polynucleotide of the present invention.

Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular, fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 mg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked DNA constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such as viral sequences, viral particles, liposome formulations, LIPOFECTIN™, precipitating agents, etc. Such methods of delivery are known in the art.

In certain embodiments, the polynucleotide constructs are complexed in a liposome preparation. Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because a tight charge complex can be formed between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which is herein incorporated by reference); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is herein incorporated by reference), in functional form.

Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are particularly useful and are available under the trademark LIPOFECTIN™, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporated by reference). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (BOEHRINGER™)

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g. PCT Publication No. WO 90/11092 (which is herein incorporated by reference) for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparation of DOTMA liposomes is explained in the literature, see, e.g., P. Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is herein incorporated by reference. Similar methods can be used to prepare liposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidyl ethanolamine (DOPE) can be used in various combinations to make conventional liposomes, with or without the addition of cholesterol. Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonication vial. The sample is placed under a vacuum pump overnight and is hydrated the following day with deionized water. The sample is then sonicated for 2 hours in a capped vial, using a Heat Systems model 350 sonicator equipped with an inverted cup (bath type) probe at the maximum setting while the bath is circulated at 15EC. Alternatively, negatively charged vesicles can be prepared without sonication to produce multilamellar vesicles or by extrusion through nucleopore membranes to produce unilamellar vesicles of discrete size. Other methods are known and available to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), with SUVs being preferred. The various liposome-nucleic acid complexes are prepared using methods well known in the art. See, e.g., Straubinger et al., Methods of Immunology (1983), 101:512-527, which is herein incorporated by reference. For example, MLVs containing nucleic acid can be prepared by depositing a thin film of phospholipid on the walls of a glass tube and subsequently hydrating with a solution of the material to be encapsulated. SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar liposomes. The material to be entrapped is added to a suspension of preformed MLVs and then sonicated. When using liposomes containing cationic lipids, the dried lipid film is resuspended in an appropriate solution such as sterile water or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated, and then the preformed liposomes are mixed directly with the DNA. The liposome and DNA form a very stable complex due to binding of the positively charged liposomes to the cationic DNA. SUVs find use with small nucleic acid fragments. LUVs are prepared by a number of methods, well known in the art. Commonly used methods include Ca²⁺-EDTA chelation (Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell 17:77 (1979)); ether injection (Deamer, D. and Bangham, A., Biochim. Biophys. Acta 443:629 (1976); Ostro et al., Biochem. Biophys. Res. Commun. 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA 76:3348 (1979)); detergent dialysis (Enoch, H. and Strittmatter, P., Proc. Natl. Acad. Sci. USA 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley et al., J. Biol. Chem. 255:10431 (1980); Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75:145 (1978); Schaefer-Ridder et al., Science 215:166 (1982)), which are herein incorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 to about 1:10. Preferably, the ration will be from about 5:1 to about 1:5. More preferably, the ration will be about 3:1 to about 1:3. Still more preferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference) reports on the injection of genetic material, complexed with cationic liposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 (which are herein incorporated by reference) provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and international publication no. WO 94/9469 provide methods for delivering DNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, using a retroviral particle containing RNA which comprises a sequence encoding a polypeptide of the present invention. Retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO₄ precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a polypeptide of the present invention. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a polypeptide of the present invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo, with polynucleotide contained in an adenovirus vector. Adenovirus can be manipulated such that it encodes and expresses a polypeptide of the present invention, and at the same time is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, thereby alleviating concerns about insertional mutagenesis. Furthermore, adenoviruses have been used as live enteric vaccines for many years with an excellent safety profile (Schwartz et al. Am. Rev. Respir. Dis.109:233-238 (1974)). Finally, adenovirus mediated gene transfer has been demonstrated in a number of instances including transfer of alpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M. A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155). Furthermore, extensive studies to attempt to establish adenovirus as a causative agent in human cancer were uniformly negative (Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

Suitable adenoviral vectors useful in the present invention are described, for example, in Kozarsky and Wilson, Curr. Opin. Genet. Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992); Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al., Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692 (1993); and U.S. Pat. No. 5,652,224, which are herein incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 region of adenovirus and constitutively express E1a and E1b, which complement the defective adenoviruses by providing the products of the genes deleted from the vector. In addition to Ad2, other varieties of adenovirus (e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention are replication deficient. Replication deficient adenoviruses require the aid of a helper virus and/or packaging cell line to form infectious particles. The resulting virus is capable of infecting cells and can express a polynucleotide of interest that is operably linked to a promoter, but cannot replicate in most cells. Replication deficient adenoviruses may be deleted in one or more of all or a portion of the following genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or in vivo, using an adeno-associated virus (AAV). AAVs are naturally occurring defective viruses that require helper viruses to produce infectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158:97 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods for producing and using such AAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present invention will include all the sequences necessary for DNA replication, encapsidation, and host-cell integration. The polynucleotide construct is inserted into the AAV vector using standard cloning methods, such as those found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAV vector is then transfected into packaging cells which are infected with a helper virus, using any standard technique, including lipofection, electroporation, calcium phosphate precipitation, etc. Appropriate helper viruses include adenoviruses, cytomegaloviruses, vaccinia viruses, or herpes viruses. Once the packaging cells are transfected and infected, they will produce infectious AAV viral particles which contain the polynucleotide construct. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo. The transduced cells will contain the polynucleotide construct integrated into its genome, and will express a polypeptide of the invention.

Another method of gene therapy involves operably associating heterologous control regions and endogenous polynucleotide sequences (e.g. encoding a polypeptide of the present invention) via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No. WO 96/29411, published Sep. 26, 1996; International Publication No. WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), which are herein encorporated by reference. This method involves the activation of a gene which is present in the target cells, but which is not normally expressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known in the art, which contain the promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to an endogenous sequence to permit homologous recombination of the promoter-targeting sequence with the endogenous sequence. The targeting sequence will be sufficiently near the 5′ end of the desired endogenous polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination.

The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter. The amplified promoter and targeting sequences are digested and ligated together.

The promoter-targeting sequence construct is delivered to the cells, either as naked polynucleotide, or in conjunction with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitating agents, etc., described in more detail above. The P promoter-targeting sequence can be delivered by any method, included direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells. Homologous recombination between the construct and the endogenous sequence takes place, such that an endogenous sequence is placed under the control of the promoter. The promoter then drives the expression of the endogenous sequence.

The polynucleotide encoding a polypeptide of the present invention may contain a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is positioned in the coding region of the polynucleotide to be expressed towards or at the 5′ end of the coding region. The signal sequence may be homologous or heterologous to the polynucleotide of interest and may be homologous or heterologous to the cells to be transfected. Additionally, the signal sequence may be chemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotides constructs can be used so long as the mode results in the expression of one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter infusion, BIOLISTIC™ injectors, particle accelerators (i.e., “gene guns”), gelfoam sponge depots, other commercially available depot materials, osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, and decanting or topical applications during surgery. For example, direct injection of naked calcium phosphate-precipitated plasmid into rat liver and rat spleen or a protein-coated plasmid into the portal vein has resulted in gene expression of the foreign gene in the rat livers (Kaneda et al., Science 243:375 (1989)).

A preferred method of local administration is by direct injection. Preferably, a recombinant molecule of the present invention complexed with a delivery vehicle is administered by direct injection into or locally within the area of arteries. Administration of a composition locally within the area of arteries refers to injecting the composition centimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotide construct of the present invention in or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the surface of tissue inside the wound or the construct can be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration include recombinant molecules of the present invention complexed to a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use with systemic administration comprise liposomes comprising ligands for targeting the vehicle to a particular site. In specific embodiments, suitable delivery vehicles for use with systemic administration comprise liposomes comprising polypeptides of the invention for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenous injection, aerosol, oral and percutaneous (topical) delivery. Intravenous injections can be performed using methods standard in the art. Aerosol delivery can also be performed using methods standard in the art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA 189:11277-11281, 1992, which is incorporated herein by reference). Oral delivery can be performed by complexing a polynucleotide construct of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers, include plastic capsules or tablets, such as those known in the art. Topical delivery can be performed by mixing a polynucleotide construct of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.

Determining an effective amount of substance to be delivered can depend upon a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, the precise condition requiring treatment and its severity, and the route of administration. The frequency of treatments depends upon a number of factors, such as the amount of polynucleotide constructs administered per dose, as well as the health and history of the subject. The precise amount, number of doses, and timing of doses will be determined by the attending physician or veterinarian.

Therapeutic compositions of the present invention can be administered to any animal, preferably to mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats, rabbits, sheep, cattle, horses and pigs, with humans being particularly preferred.

Biological Activities

Polynucleotides or polypeptides, or agonists or antagonists of the present invention, can be used in assays to test for one or more biological activities. If these polynucleotides or polypeptides, or agonists or antagonists of the present invention, do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides, and agonists or antagonists could be used to treat the associated disease.

Members of the secreted family of proteins are believed to be involved in biological activities associated with, for example, cellular signaling. Accordingly, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in diagnosis, prognosis, prevention and/or treatment of diseases and/or disorders associated with aberrant activity of secreted polypeptides.

In preferred embodiments, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, prognosis, prevention, treatment, and/or amelioration of diseases and/or disorders relating to the endocrine system, the nervous system (See, for example, “Neurological Disorders” section below), the immune system (See, for example, “Immune Activity” section below), the gastrointestinal system (e.g., Crohn's disease, pancreatitis, gallstones, antibiotic-associated colitis, duodenitis, gastrointestinal neoplasms, and as described in the “Gastrointestinal Disorders” section below), or the cardiovascular system (e.g., atherosclerosis, stroke, myocardial infarction, hypertension, and as described in the “Cardiovascular Disorders” section below). In preferred embodiments, compositions of the invention (including polynucleotides, polypeptides and antibodies of the invention, and fragments and variants thereof) may be used in the diagnosis, prognosis, prevention, treatment, and/or amelioration of cancer and other hyperproliferative diseases and/or disorders (e.g., as described in the “Hyperproliferative Disorders”).

In certain embodiments, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognosticate diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed including one, two, three, four, five, or more tissues disclosed in Table 1B.2 (such as column 5) (Tissue Distribution Library Code).

Thus, polynucleotides, translation products and antibodies of the invention are useful in the diagnosis, detection, prevention, prognistication, and/or treatment of diseases and/or disorders associated with activities that include, but are not limited to, prohormone activation, neurotransmitter activity, cellular signaling, cellular proliferation, cellular differentiation, and cell migration.

More generally, polynucleotides, translation products and antibodies corresponding to this gene may be useful for the diagnosis, prognosis, prevention, treatment and/or amelioration of diseases and/or disorders associated with the following system or systems.

Immune Activity

Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases, disorders, and/or conditions of the immune system, by, for example, activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells. Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells. The etiology of these immune diseases, disorders, and/or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular immune system disease or disorder.

In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to treat diseases and disorders of the immune system and/or to inhibit or enhance an immune response generated by cells associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).

Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating or detecting deficiencies or disorders of hematopoietic cells. Polynucleotides or polypeptides, or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat and/or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells.

Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in preventing, diagnosing, prognosticating, treating and/or ameliorating immunodeficiencies, including both congenital and acquired immunodeficiencies. Examples of immunologic deficiency syndromes include, but are not limited to: blood protein disorders, ataxia telangiectasia, Digeorge Syndrome, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria. Examples of B cell immunodeficiencies in which immunoglobulin levels B cell function and/or B cell numbers are decreased include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia including congenital and acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type), Selective IgM deficiency, selective IgA deficiency, selective IgG subclass deficiencies, IgG subclass deficiency (with or without IgA deficiency), Ig deficiency with increased IgM, IgG and IgA deficiency with increased IgM, antibody deficiency with normal or elevated Igs, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), common variable immunodeficiency (CVID), common variable immunodeficiency (CVI) (acquired), and transient hypogammaglobulinemia of infancy.

In specific embodiments, ataxia-telangiectasia or conditions associated with ataxia-telangiectasia are detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated using the polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof.

Examples of congenital immunodeficiencies in which T cell and/or B cell function and/or number is decreased include, but are not limited to: DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including, but not limited to, X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2 deletion, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4+ T-lymphocytopenia, immunodeficiency with predominant T cell defect (unspecified), and unspecified immunodeficiency of cell mediated immunity.

In specific embodiments, DiGeorge anomaly or conditions associated with DiGeorge anomaly are prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polypeptides or polynucleotides of the invention, or antagonists or agonists thereof.

Other immunodeficiencies that may be prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, include, but are not limited to, chronic granulomatous disease, Chédiak-Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6-phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, complement component deficiencies (including C1, C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphoplasia-aplasia, immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short limbed dwarfism, and Nezelof syndrome-combined immunodeficiency with Igs.

In a preferred embodiment, the immunodeficiencies and/or conditions associated with the immunodeficiencies recited above are prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.

In a preferred embodiment polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals. In specific embodiments, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among B cell and/or T cell immunodeficient individuals.

The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in preventing, detecting, diagnosing, prognosticating, treating and/or ameliorating autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of polynucleotides and polypeptides of the invention that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.

Autoimmune diseases or disorders that may be prevented, detected, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, one or more of the following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, hypothyroidism, Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, purpura (e.g., Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), inflammatory myopathies, insulin resistance, and insulin-resistant diabetes mellitus.

Additional disorders that are likely to have an autoimmune component that may be prevented, detected, diagnosed, prognosticated, treated and/or ameliorated with the compositions of the invention include, but are not limited to, type II collagen-induced arthritis, Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Grave's Disease, multiple sclerosis, myasthenia gravis, neuritis, ophthalmia, bullous pemphigoid, pemphigus, polyendocrinopathies, purpura, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus (SLE), autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, and autoimmune inflammatory eye disorders.

Additional disorders that are likely to have an autoimmune component that may be prevented, detected, diagnosed, prognosticated, treated and/or ameliorated with the compositions of the invention include, but are not limited to, scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g., by glomerular basement membrane antibodies or immune complexes), bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjogren's syndrome (often characterized, e.g., by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes mellitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).

Additional disorders that may have an autoimmune component that may be prevented, detected, diagnosed, prognosticated, treated and/or ameliorated with the compositions of the invention include, but are not limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitochondria antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to IgE), asthma (often characterized, e.g., by IgG and IgM antibodies to IgE), and many other inflammatory, granulomatous, degenerative, and atrophic disorders.

In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using for example, antagonists or agonists, polypeptides or polynucleotides, or antibodies of the present invention. In a specific preferred embodiment, rheumatoid arthritis is prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.

In another specific preferred embodiment, systemic lupus erythematosus is prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention. In another specific preferred embodiment, idiopathic thrombocytopenia purpura is prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.

In another specific preferred embodiment IgA nephropathy is prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention.

In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are prevented, detected, diagnosed, prognosticated, treated and/or ameliorated using polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention

In preferred embodiments, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a immunosuppressive agent(s).

Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases, disorders, and/or conditions of hematopoietic cells. Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with a decrease in certain (or many) types hematopoietic cells, including but not limited to, leukopenia, neutropenia, anemia, and thrombocytopenia. Alternatively, Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat or prevent those diseases, disorders, and/or conditions associated with an increase in certain (or many) types of hematopoietic cells, including but not limited to, histiocytosis.

Allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated using polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof. Moreover, these molecules can be used to treat, prevent, prognose, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.

Additionally, polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhinitis, and eczema. In specific embodiments, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to modulate IgE concentrations in vitro or in vivo.

Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention have uses in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of inflammatory conditions. For example, since polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists of the invention may inhibit the activation, proliferation and/or differentiation of cells involved in an inflammatory response, these molecules can be used to prevent and/or treat chronic and acute inflammatory conditions. Such inflammatory conditions include, but are not limited to, for example, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome), ischemia-reperfusion injury, endotoxin lethality, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, over production of cytokines (e.g., TNF or IL-1.), respiratory disorders (e.g., asthma and allergy); gastrointestinal disorders (e.g., inflammatory bowel disease); cancers (e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders (e.g., multiple sclerosis; blood brain permeability, ischemic brain injury and/or stroke, traumatic brain injury, neurodegenerative disorders (e.g., Parkinson's disease and Alzheimer's disease); AIDS-related dementia; and prion disease); cardiovascular disorders (e.g., atherosclerosis, myocarditis, cardiovascular disease, and cardiopulmonary bypass complications); as well as many additional diseases, conditions, and disorders that are characterized by inflammation (e.g., hepatitis, rheumatoid arthritis, gout, trauma, septic shock, pancreatitis, sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave's disease, systemic lupus erythematosus, diabetes mellitus, and allogenic transplant rejection).

Because inflammation is a fundamental defense mechanism, inflammatory disorders can effect virtually any tissue of the body. Accordingly, polynucleotides, polypeptides, and antibodies of the invention, as well as agonists or antagonists thereof, have uses in the treatment of tissue-specific inflammatory disorders, including, but not limited to, adrenalitis, alveolitis, angiocholecystitis, appendicitis, balanitis, blepharitis, bronchitis, bursitis, carditis, cellulitis, cervicitis, cholecystitis, chorditis, cochlitis, colitis, conjunctivitis, cystitis, dermatitis, diverticulitis, encephalitis, endocarditis, esophagitis, eustachitis, fibrositis, folliculitis, gastritis, gastroenteritis, gingivitis, glossitis, hepatosplenitis, keratitis, labyrinthitis, laryngitis, lymphangitis, mastitis, media otitis, meningitis, metritis, mucitis, myocarditis, myosititis, myringitis, nephritis, neuritis, orchitis, osteochondritis, otitis, pericarditis, peritendonitis, peritonitis, pharyngitis, phlebitis, poliomyelitis, prostatitis, pulpitis, retinitis, rhinitis, salpingitis, scleritis, sclerochoroiditis, scrotitis, sinusitis, spondylitis, steatitis, stomatitis, synovitis, syringitis, tendonitis, tonsillitis, urethritis, and vaginitis.

In specific embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate organ transplant rejections and graft-versus-host disease (GVHD). Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. Polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD. In specific embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, that inhibit an immune response, particularly the activation, proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing experimental allergic and hyperacute xenograft rejection.

In specific embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to diagnose, prognose, prevent, and/or treat, autoimmune and inflammatory diseases (e.g., immune complex-induced vasculitis, glomerulonephritis, hemolytic anemia, myasthenia gravis, type II collagen-induced arthritis, experimental allergic and hyperacute xenograft rejection, rheumatoid arthritis, and systemic lupus erythematosus (SLE).

Similarly, a polynucleotides or polypeptides, or agonists or antagonists of the present invention may also be used to modulate and/or diagnose inflammation. For example, the polypeptide or polynucleotide or agonists or antagonist may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat, prevent, diagnose and/or prognose inflammatory conditions, both chronic and acute conditions, including, but not limited to, chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)

In other embodiments, polypeptides, antibodies, or polynucleotides of the invention, and/or agonists or antagonists thereof, are useful to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate immune complex diseases, including, but not limited to, serum sickness, post streptococcal glomerulonephritis, polyarteritis nodosa, and immune complex-induced vasculitis.

Polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the invention can be used to treat, detect, and/or prevent infectious agents. For example, by increasing the immune response, particularly increasing the proliferation activation and/or differentiation of B and/or T cells, infectious diseases may be treated, detected, and/or prevented. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may also directly inhibit the infectious agent (refer to section of application listing infectious agents, etc), without necessarily eliciting an immune response.

In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a vaccine adjuvant that enhances immune responsiveness to an antigen. In a specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance tumor-specific immune responses.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include virus and virus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, respiratory syncytial virus, Dengue, rotavirus, Japanese B encephalitis, influenza A and B, parainfluenza, measles, cytomegalovirus, rabies, Junin, Chikungunya, Rift Valley Fever, herpes simplex, and yellow fever.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B.

In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, Plasmodium (malaria), and Borrelia burgdorferi.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria) or Leishmania.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed to treat infectious diseases including silicosis, sarcoidosis, and idiopathic pulmonary fibrosis; for example, by preventing the recruitment and activation of mononuclear phagocytes.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an antigen for the generation of antibodies to inhibit or enhance immune mediated responses against polypeptides of the invention.

In one embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are administered to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production and immunoglobulin class switching (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a stimulator of B cell responsiveness to pathogens.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an activator of T cells.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to induce higher affinity antibodies.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to increase serum immunoglobulin concentrations.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to accelerate recovery of immunocompromised individuals.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among aged populations and/or neonates.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, prior to the beginning of recovery of T-cell populations. In another specific embodiment, compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function. Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency. Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the polypeptides, antibodies, polynucleotides and/or agonists or antagonists thereof, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, and recovery from surgery.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a regulator of antigen presentation by monocytes, dendritic cells, and/or B-cells. In one embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention enhance antigen presentation or antagonizes antigen presentation in vitro or in vivo. Moreover, in related embodiments, said enhancement or antagonism of antigen presentation may be useful as an anti-tumor treatment or to modulate the immune system.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an agent to direct an individual's immune system towards development of a humoral response (i.e. TH2) as opposed to a TH1 cellular response.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a stimulator of B cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable Immunodificiency.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect. In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used in the pretreatment of bone marrow samples prior to transplant.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a gene-based therapy for genetically inherited disorders resulting in immuno-incompetence/immunodeficiency such as observed among SCID patients.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leishmania.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of regulating secreted cytokines that are elicited by polypeptides of the invention.

In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used in one or more of the applications described herein, as they may apply to veterinary medicine.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of blocking various aspects of immune responses to foreign agents or self. Examples of diseases or conditions in which blocking of certain aspects of immune responses may be desired include autoimmune disorders such as lupus, and arthritis, as well as immunoresponsiveness to skin allergies, inflammation, bowel disease, injury and diseases/disorders associated with pathogens.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for preventing the B cell proliferation and Ig secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and multiple sclerosis.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a inhibitor of B and/or T cell migration in endothelial cells. This activity disrupts tissue architecture or cognate responses and is useful, for example in disrupting immune responses, and blocking sepsis.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for chronic hypergammaglobulinemia evident in such diseases as monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monoclonal gammopathies, and plasmacytomas.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed for instance to inhibit polypeptide chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells, in certain autoimmune and chronic inflammatory and infective diseases. Examples of autoimmune diseases are described herein and include multiple sclerosis, and insulin-dependent diabetes.

The polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed to treat idiopathic hyper-eosinophilic syndrome by, for example, preventing eosinophil production and migration.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used to enhance or inhibit complement mediated cell lysis.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used to enhance or inhibit antibody dependent cellular cytotoxicity.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may also be employed for treating atherosclerosis, for example, by preventing monocyte infiltration in the artery wall.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed to treat adult respiratory distress syndrome (ARDS).

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be useful for stimulating wound and tissue repair, stimulating angiogenesis, and/or stimulating the repair of vascular or lymphatic diseases or disorders. Additionally, agonists and antagonists of the invention may be used to stimulate the regeneration of mucosal surfaces.

In a specific embodiment, polynucleotides or polypeptides, and/or agonists thereof are used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate a disorder characterized by primary or acquired immunodeficiency, deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, polynucleotides or polypeptides, and/or agonists thereof may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pneumocystis carnii. Other diseases and disorders that may be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with polynucleotides or polypeptides, and/or agonists of the present invention include, but are not limited to, HIV infection, HTLV-BLV infection, lymphopenia, phagocyte bactericidal dysfunction anemia, thrombocytopenia, and hemoglobinuria.

In another embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention are used to treat, and/or diagnose an individual having common variable immunodeficiency disease (“CVID”; also known as “acquired agammaglobulinemia” and “acquired hypogammaglobulinemia”) or a subset of this disease.

In a specific embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate cancers or neoplasms including immune cell or immune tissue-related cancers or neoplasms. Examples of cancers or neoplasms that may be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, acute myelogenous leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL) Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, EBV-transformed diseases, and/or diseases and disorders described in the section entitled “Hyperproliferative Disorders” elsewhere herein.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of activating T cells.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as an inhibitor of graft versus host disease or transplant rejection.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for B cell and/or T cell malignancies such as ALL, Hodgkins disease, non-Hodgkins lymphoma, Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, and EBV-transformed diseases.

In another embodiment, administration of polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the invention, may be used to treat or prevent IgE-mediated allergic reactions including, but not limited to, asthma, rhinitis, and eczema.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a therapy for decreasing cellular proliferation of Large B-cell Lymphomas.

In another specific embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are used as a means of decreasing the involvement of B cells and Ig associated with Chronic Myelogenous Leukemia.

In specific embodiments, the compositions of the invention are used as an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy.

Antagonists of the invention include, for example, binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes or soluble forms of the polypeptides of the present invention (e.g., Fc fusion protein; see, e.g., Example 9). Agonists of the invention include, for example, binding or stimulatory antibodies, and soluble forms of the polypeptides (e.g., Fc fusion proteins; see, e.g., Example 9). polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described herein.

In another embodiment, polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention are administered to an animal (including, but not limited to, those listed above, and also including transgenic animals) incapable of producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobulin molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. WO98/24893, WO/9634096, WO/9633735, and WO/9110741). Administration of polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention to such animals is useful for the generation of monoclonal antibodies against the polypeptides, antibodies, polynucleotides and/or agonists or antagonists of the present invention.

Blood-Related Disorders

The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to modulate hemostatic (the stopping of bleeding) or thrombolytic (clot dissolving) activity. For example, by increasing hemostatic or thrombolytic activity, polynucleotides or polypeptides, and/or agonists or antagonists of the present invention could be used to treat or prevent blood coagulation diseases, disorders, and/or conditions (e.g., afibrinogenemia, factor deficiencies, hemophilia), blood platelet diseases, disorders, and/or conditions (e.g., thrombocytopenia), or wounds resulting from trauma, surgery, or other causes. Alternatively, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment or prevention of heart attacks (infarction), strokes, or scarring.

In specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention, include, but are not limited to, the prevention of occlusions in extrcorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).

In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate diseases and disorders of the blood and/or blood forming organs associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).

The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to modulate hematopoietic activity (the formation of blood cells). For example, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to increase the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of anemias and leukopenias described below. Alternatively, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to decrease the quantity of all or subsets of blood cells, such as, for example, erythrocytes, lymphocytes (B or T cells), myeloid cells (e.g., basophils, eosinophils, neutrophils, mast cells, macrophages) and platelets. The ability to decrease the quantity of blood cells or subsets of blood cells may be useful in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of leukocytoses, such as, for example eosinophilia.

The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate blood dyscrasia.

Anemias are conditions in which the number of red blood cells or amount of hemoglobin (the protein that carries oxygen) in them is below normal. Anemia may be caused by excessive bleeding, decreased red blood cell production, or increased red blood cell destruction (hemolysis). The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating anemias. Anemias that may be treated detect, prevented, diagnosed, prognosticated, treated, and/or ameliorated by the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include iron deficiency anemia, hypochromic anemia, microcytic anemia, chlorosis, hereditary sideroblastic anemia, idiopathic acquired sideroblastic anemia, red cell aplasia, megaloblastic anemia (e.g., pernicious anemia, (vitamin B12 deficiency) and folic acid deficiency anemia), aplastic anemia, hemolytic anemias (e.g., autoimmune helolytic anemia, microangiopathic hemolytic anemia, and paroxysmal nocturnal hemoglobinuria). The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating anemias associated with diseases including but not limited to, anemias associated with systemic lupus erythematosus, cancers, lymphomas, chronic renal disease, and enlarged spleens. The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating anemias arising from drug treatments such as anemias associated with methyldopa, dapsone, and/or sulfadrugs. Additionally, rhe polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating anemias associated with abnormal red blood cell architecture including but not limited to, hereditary spherocytosis, hereditary elliptocytosis, glucose-6-phosphate dehydrogenase deficiency, and sickle cell anemia.

The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating hemoglobin abnormalities, (e.g., those associated with sickle cell anemia, hemoglobin C disease, hemoglobin S—C disease, and hemoglobin E disease). Additionally, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating thalassemias, including, but not limited to major and minor forms of alpha-thalassemia and beta-thalassemia.

In another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating bleeding disorders including, but not limited to, thrombocytopenia (e.g., idiopathic thrombocytopenic purpura, and thrombotic thrombocytopenic purpura), Von Willebrand's disease, hereditary platelet disorders (e.g., storage pool disease such as Chediak-Higashi and Hermansky-Pudlak syndromes, thromboxane A2 dysfunction, thromboasthenia, and Bernard-Soulier syndrome), hemolytic-uremic syndrome, hemophelias such as hemophelia A or Factor VII deficiency and Christmas disease or Factor IX deficiency, Hereditary Hemorhhagic Telangiectsia, also known as Rendu-Osler-Weber syndrome, allergic purpura (Henoch Schonlein purpura) and disseminated intravascular coagulation.

The effect of the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention on the clotting time of blood may be monitored using any of the clotting tests known in the art including, but not limited to, whole blood partial thromboplastin time (PTT), the activated partial thromboplastin time (aPTT), the activated clotting time (ACT), the recalcified activated clotting time, or the Lee-White Clotting time.

Several diseases and a variety of drugs can cause platelet dysfunction. Thus, in a specific embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating acquired platelet dysfunction such as platelet dysfunction accompanying kidney failure, leukemia, multiple myeloma, cirrhosis of the liver, and systemic lupus erythematosus as well as platelet dysfunction associated with drug treatments, including treatment with aspirin, ticlopidine, nonsteroidal anti-inflammatory drugs (used for arthritis, pain, and sprains), and penicillin in high doses.

In another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders characterized by or associated with increased or decreased numbers of white blood cells. Leukopenia occurs when the number of white blood cells decreases below normal. Leukopenias include, but are not limited to, neutropenia and lymphocytopenia. An increase in the number of white blood cells compared to normal is known as leukocytosis. The body generates increased numbers of white blood cells during infection. Thus, leukocytosis may simply be a normal physiological parameter that reflects infection. Alternatively, leukocytosis may be an indicator of injury or other disease such as cancer. Leokocytoses, include but are not limited to, eosinophilia, and accumulations of macrophages. In specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating leukopenia. In other specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating leukocytosis.

Leukopenia may be a generalized decreased in all types of white blood cells, or may be a specific depletion of particular types of white blood cells. Thus, in specific embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating decreases in neutrophil numbers, known as neutropenia. Neutropenias that may be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, infantile genetic agranulocytosis, familial neutropenia, cyclic neutropenia, neutropenias resulting from or associated with dietary deficiencies (e.g., vitamin B 12 deficiency or folic acid deficiency), neutropenias resulting from or associated with drug treatments (e.g., antibiotic regimens such as penicillin treatment, sulfonamide treatment, anticoagulant treatment, anticonvulsant drugs, anti-thyroid drugs, and cancer chemotherapy), and neutropenias resulting from increased neutrophil destruction that may occur in association with some bacterial or viral infections, allergic disorders, autoimmune diseases, conditions in which an individual has an enlarged spleen (e.g., Felty syndrome, malaria and sarcoidosis), and some drug treatment regimens.

The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating lymphocytopenias (decreased numbers of B and/or T lymphocytes), including, but not limited lymphocytopenias resulting from or associated with stress, drug treatments (e.g., drug treatment with corticosteroids, cancer chemotherapies, and/or radiation therapies), AIDS infection and/or other diseases such as, for example, cancer, rheumatoid arthritis, systemic lupus erythematosus, chronic infections, some viral infections and/or hereditary disorders (e.g., DiGeorge syndrome, Wiskott-Aldrich Syndome, severe combined immunodeficiency, ataxia telangiectsia).

The polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders associated with macrophage numbers and/or macrophage function including, but not limited to, Gaucher's disease, Niemann-Pick disease, Letterer-Siwe disease and Hand-Schuller-Christian disease.

In another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders associated with eosinophil numbers and/or eosinophil function including, but not limited to, idiopathic hypereosinophilic syndrome, eosinophilia-myalgia syndrome, and Hand-Schuller-Christian disease.

In yet another embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating leukemias and lymphomas including, but not limited to, acute lymphocytic (lymphpblastic) leukemia (ALL), acute myeloid (myelocytic, myelogenous, myeloblastic, or myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., B cell leukemias, T cell leukemias, Sezary syndrome, and Hairy cell leukenia), chronic myelocytic (myeloid, myelogenous, or granulocytic) leukemia, Hodgkin's lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, and mycosis fungoides.

In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases and disorders of plasma cells including, but not limited to, plasma cell dyscrasias, monoclonal gammaopathies, monoclonal gammopathies of undetermined significance, multiple myeloma, macroglobulinemia, Waldenstrom's macroglobulinemia, cryoglobulinemia, and Raynaud's phenomenon.

In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating myeloproliferative disorders, including but not limited to, polycythemia vera, relative polycythemia, secondary polycythemia, myelofibrosis, acute myelofibrosis, agnogenic myelod metaplasia, thrombocythemia, (including both primary and seconday thrombocythemia) and chronic myelocytic leukemia.

In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as a treatment prior to surgery, to increase blood cell production.

In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to enhance the migration, phagocytosis, superoxide production, antibody dependent cellular cytotoxicity of neutrophils, eosionophils and macrophages.

In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to increase the number of stem cells in circulation prior to stem cells pheresis. In another specific embodiment, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to increase the number of stem cells in circulation prior to platelet pheresis.

In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful as an agent to increase cytokine production.

In other embodiments, the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating primary hematopoietic disorders.

Hyperproliferative Disorders

In certain embodiments, polynucleotides or polypeptides, or agonists or antagonists of the present invention can be used to treat or detect hyperproliferative disorders, including neoplasms. Polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions. Alternatively, Polynucleotides or polypeptides, or agonists or antagonists of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasing antigenic qualities of the hyperproliferative disorder or by proliferating, differentiating, or mobilizing T-cells, hyperproliferative disorders can be treated. This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, decreasing an immune response may also be a method of treating hyperproliferative disorders, such as a chemotherapeutic agent.

Examples of hyperproliferative disorders that can be treated or detected by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.

Similarly, other hyperproliferative disorders can also be treated or detected by polynucleotides or polypeptides, or agonists or antagonists of the present invention. Examples of such hyperproliferative disorders include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Disease, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma/Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/Malignant Fibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma, Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.

In another preferred embodiment, polynucleotides or polypeptides, or agonists or antagonists of the present invention are used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate premalignant conditions and to prevent progression to a neoplastic or malignant state, including but not limited to those disorders described above. Such uses are indicated in conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-79.)

Hyperplasia is a form of controlled cell proliferation, involving an increase in cell number in a tissue or organ, without significant alteration in structure or function. Hyperplastic disorders which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, angiofollicular mediastinal lymph node hyperplasia, angiolymphoid hyperplasia with eosinophilia, atypical melanocytic hyperplasia, basal cell hyperplasia, benign giant lymph node hyperplasia, cementum hyperplasia, congenital adrenal hyperplasia, congenital sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasia of the breast, denture hyperplasia, ductal hyperplasia, endometrial hyperplasia, fibromuscular hyperplasia, focal epithelial hyperplasia, gingival hyperplasia, inflammatory fibrous hyperplasia, inflammatory papillary hyperplasia, intravascular papillary endothelial hyperplasia, nodular hyperplasia of prostate, nodular regenerative hyperplasia, pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia, and verrucous hyperplasia.

Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplastic disorders which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, agnogenic myeloid metaplasia, apocrine metaplasia, atypical metaplasia, autoparenchymatous metaplasia, connective tissue metaplasia, epithelial metaplasia, intestinal metaplasia, metaplastic anemia, metaplastic ossification, metaplastic polyps, myeloid metaplasia, primary myeloid metaplasia, secondary myeloid metaplasia, squamous metaplasia, squamous metaplasia of amnion, and symptomatic myeloid metaplasia.

Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation. Dysplastic disorders which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia, cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, craniodiaphysial dysplasia, craniocarpotarsal dysplasia, craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia, ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia, dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex, dysplasia epiphysialis punctata, epithelial dysplasia, faciodigitogenital dysplasia, familial fibrous dysplasia of jaws, familial white folded dysplasia, fibromuscular dysplasia, fibrous dysplasia of bone, florid osseous dysplasia, hereditary renal-retinal dysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermal dysplasia, lymphopenic thymic dysplasia, mammary dysplasia, mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia, monostotic fibrous dysplasia, mucoepithelial dysplasia, multiple epiphysial dysplasia, oculoauriculovertebral dysplasia, oculodentodigital dysplasia, oculovertebral dysplasia, odontogenic dysplasia, ophthalmomandibulomelic dysplasia, periapical cemental dysplasia, polyostotic fibrous dysplasia, pseudoachondroplastic spondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia, spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention (including polynucleotides, polypeptides, agonists or antagonists) include, but are not limited to, benign dysproliferative disorders (e.g., benign tumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps, colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen's disease, Farmer's Skin, solar cheilitis, and solar keratosis.

In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to diagnose and/or prognosticate disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).

In another embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat cancers and neoplasms, including, but not limited to those described herein. In a further preferred embodiment, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention conjugated to a toxin or a radioactive isotope, as described herein, may be used to treat acute myelogenous leukemia.

Additionally, polynucleotides, polypeptides, and/or agonists or antagonists of the invention may affect apoptosis, and therefore, would be useful in treating a number of diseases associated with increased cell survival or the inhibition of apoptosis. For example, diseases associated with increased cell survival or the inhibition of apoptosis that could be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.

In preferred embodiments, polynucleotides, polypeptides, and/or agonists or antagonists of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cell survival that could be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis that could be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

Hyperproliferative diseases and/or disorders that could be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention, include, but are not limited to, neoplasms located in the liver, abdomen, bone, breast, digestive system, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thorax, and urogenital tract.

Similarly, other hyperproliferative disorders can also be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by polynucleotides, polypeptides, and/or agonists or antagonists of the invention. Examples of such hyperproliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.

Another preferred embodiment utilizes polynucleotides of the present invention to inhibit aberrant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein said polynucleotide represses said expression.

Another embodiment of the present invention provides a method of treating cell-proliferative disorders in individuals comprising administration of one or more active gene copies of the present invention to an abnormally proliferating cell or cells. In a preferred embodiment, polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides. In another preferred embodiment of the present invention, the DNA construct encoding the polynucleotides of the present invention is inserted into cells to be treated utilizing a retrovirus, or more preferably an adenoviral vector (See G J. Nabel, et. al., PNAS 1999 96: 324-326, which is hereby incorporated by reference). In a most preferred embodiment, the viral vector is defective and will not transform non-proliferating cells, only proliferating cells. Moreover, in a preferred embodiment, the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides, can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product. As such the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens. By “repressing expression of the oncogenic genes” is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre-message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.

For local administration to abnormally proliferating cells, polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, LIPOFECTIN™, or as naked polynucleotides, or any other method described throughout the specification. The polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad. Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol. Cell. Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yates et al., Nature 313:812 (1985)) known to those skilled in the art. These references are exemplary only and are hereby incorporated by reference. In order to specifically deliver or transfect cells which are abnormally proliferating and spare non-dividing cells, it is preferable to utilize a retrovirus, or adenoviral (as described in the art and elsewhere herein) delivery system known to those of skill in the art. Since host DNA replication is required for retroviral DNA to integrate and the retrovirus will be unable to self replicate due to the lack of the retrovirus genes needed for its life cycle. Utilizing such a retroviral delivery system for polynucleotides of the present invention will target said gene and constructs to abnormally proliferating cells and will spare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site. The polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant.

Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells. Moreover, it is possible to administer more than one of the polynucleotide of the present invention simultaneously to the same site. By “biologically inhibiting” is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells. The biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating one or more of the described disorders. Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnosis, prognosis, monitoring, or therapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the present invention are useful for treating a subject having or developing cell proliferative and/or differentiation disorders as described herein. Such treatment comprises administering a single or multiple doses of the antibody, or a fragment, derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors, for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10¹⁵ M, and 10⁻¹⁵ M.

Moreover, polypeptides of the present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein. In a most preferred embodiment, said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor-associated macrophages (See Joseph I B, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated by reference). Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al., Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby incorporated by reference)).

Polypeptides, including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis. Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor-1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (See Schulze-Osthoff K, et. al., Eur J Biochem 254(3):439-59 (1998), which is hereby incorporated by reference). Moreover, in another preferred embodiment of the present invention, said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins, anti-inflammatory proteins (See for example, Mutat Res 400(1-2):447-55 (1998), Med Hypotheses. 50(5):423-33 (1998), Chem Biol Interact. April 24; 111-112:23-34 (1998), J Mol Med. 76(6):402-12 (1998), Int J Tissue React; 20(1):3-15 (1998), which are all hereby incorporated by reference).

Polypeptides, including protein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewhere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol 1998; 231:125-41, which is hereby incorporated by reference). Such therapeutic effects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing polypeptides or polypeptide antibodies associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention. Polypeptides or polypeptide antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.

Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention ‘vaccinated’ the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.

Renal Disorders

Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate disorders of the renal system. Renal disorders which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention include, but are not limited to, kidney failure, nephritis, blood vessel disorders of kidney, metabolic and congenital kidney disorders, urinary disorders of the kidney, autoimmune disorders, sclerosis and necrosis, electrolyte imbalance, and kidney cancers.

Kidney diseases which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with compositions of the invention include, but are not limited to, acute kidney failure, chronic kidney failure, atheroembolic renal failure, end-stage renal disease, inflammatory diseases of the kidney (e.g., acute glomerulonephritis, postinfectious glomerulonephritis, rapidly progressive glomerulonephritis, nephrotic syndrome, membranous glomerulonephritis, familial nephrotic syndrome, membranoproliferative glomerulonephritis I and II, mesangial proliferative glomerulonephritis, chronic glomerulonephritis, acute tubulointerstitial nephritis, chronic tubulointerstitial nephritis, acute post-streptococcal glomerulonephritis (PSGN), pyelonephritis, lupus nephritis, chronic nephritis, interstitial nephritis, and post-streptococcal glomerulonephritis), blood vessel disorders of the kidneys (e.g., kidney infarction, atheroembolic kidney disease, cortical necrosis, malignant nephrosclerosis, renal vein thrombosis, renal underperfusion, renal retinopathy, renal ischemia-reperfusion, renal artery embolism, and renal artery stenosis), and kidney disorders resulting form urinary tract disease (e.g., pyelonephritis, hydronephrosis, urolithiasis (renal lithiasis, nephrolithiasis), reflux nephropathy, urinary tract infections, urinary retention, and acute or chronic unilateral obstructive uropathy.)

In addition, compositions of the invention can be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate metabolic and congenital disorders of the kidney (e.g., uremia, renal amyloidosis, renal osteodystrophy, renal tubular acidosis, renal glycosuria, nephrogenic diabetes insipidus, cystinuria, Fanconi's syndrome, renal fibrocystic osteosis (renal rickets), Hartnup disease, Bartter's syndrome, Liddle's syndrome, polycystic kidney disease, medullary cystic disease, medullary sponge kidney, Alport's syndrome, nail-patella syndrome, congenital nephrotic syndrome, CRUSH syndrome, horseshoe kidney, diabetic nephropathy, nephrogenic diabetes insipidus, analgesic nephropathy, kidney stones, and membranous nephropathy), and autoimmune disorders of the kidney (e.g., systemic lupus erythematosus (SLE), Goodpasture syndrome, IgA nephropathy, and IgM mesangial proliferative glomerulonephritis).

Compositions of the invention can also be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate sclerotic or necrotic disorders of the kidney (e.g., glomerulosclerosis, diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), necrotizing glomerulonephritis, and renal papillary necrosis), cancers of the kidney (e.g., nephroma, hypernephroma, nephroblastoma, renal cell cancer, transitional cell cancer, renal adenocarcinoma, squamous cell cancer, and Wilm's tumor), and electrolyte imbalances (e.g., nephrocalcinosis, pyuria, edema, hydronephritis, proteinuria, hyponatremia, hypernatremia, hypokalemia, hyperkalemia, hypocalcemia, hypercalcemia, hypophosphatemia, and hyperphosphatemia).

Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, BIOLISTIC™ injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides are described in more detail herein.

Cardiovascular Disorders

Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate cardiovascular diseases and disorders, including, but not limited to, peripheral artery disease, such as limb ischemia.

Cardiovascular disorders include, but are not limited to, cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include, but are not limited to, aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.

Cardiovascular disorders also include, but are not limited to, heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include, but are not limited to, sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve diseases include, but are not limited to, aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.

Myocardial diseases include, but are not limited to, alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemias include, but are not limited to, coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include, but are not limited to, dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include, but are not limited to, arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include, but are not limited to, carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.

Embolisms include, but are not limited to, air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms. Thrombosis include, but are not limited to, coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemic disorders include, but are not limited to, cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia. Vasculitis includes, but is not limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.

Polynucleotides or polypeptides, or agonists or antagonists of the invention, are especially effective for the treatment of critical limb ischemia and coronary disease.

Polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, BIOLISTIC™ injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art. Polypeptides may be administered as part of a Therapeutic, described in more detail below. Methods of delivering polynucleotides are described in more detail herein.

Respiratory Disorders

Polynucleotides or polypeptides, or agonists or antagonists of the present invention may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate diseases and/or disorders of the respiratory system.

Diseases and disorders of the respiratory system include, but are not limited to, nasal vestibulitis, nonallergic rhinitis (e.g., acute rhinitis, chronic rhinitis, atrophic rhinitis, vasomotor rhinitis), nasal polyps, and sinusitis, juvenile angiofibromas, cancer of the nose and juvenile papillomas, vocal cord polyps, nodules (singer's nodules), contact ulcers, vocal cord paralysis, laryngoceles, pharyngitis (e.g., viral and bacterial), tonsillitis, tonsillar cellulitis, parapharyngeal abscess, laryngitis, laryngoceles, and throat cancers (e.g., cancer of the nasopharynx, tonsil cancer, larynx cancer), lung cancer (e.g., squamous cell carcinoma, small cell (oat cell) carcinoma, large cell carcinoma, and adenocarcinoma), allergic disorders (eosinophilic pneumonia, hypersensitivity pneumonitis (e.g., extrinsic allergic alveolitis, allergic interstitial pneumonitis, organic dust pneumoconiosis, allergic bronchopulmonary aspergillosis, asthma, Wegener's granulomatosis (granulomatous vasculitis), Goodpasture's syndrome)), pneumonia (e.g., bacterial pneumonia (e.g., Streptococcus pneumoniae (pneumoncoccal pneumonia), Staphylococcus aureus (staphylococcal pneumonia), Gram-negative bacterial pneumonia (caused by, e.g., Klebsiella and Pseudomas spp.), Mycoplasma pneumoniae pneumonia, Hemophilus influenzae pneumonia, Legionella pneumophila (Legionnaires' disease), and Chlamydia psittaci (Psittacosis)), and viral pneumonia (e.g., influenza, chickenpox (varicella).

Additional diseases and disorders of the respiratory system include, but are not limited to bronchiolitis, polio (poliomyelitis), croup, respiratory syncytial viral infection, mumps, erythema infectiosum (fifth disease), roseola infantum, progressive rubella panencephalitis, german measles, and subacute sclerosing panencephalitis), fungal pneumonia (e.g., Histoplasmosis, Coccidioidomycosis, Blastomycosis, fungal infections in people with severely suppressed immune systems (e.g., cryptococcosis, caused by Cryptococcus neoformans; aspergillosis, caused by Aspergillus spp.; candidiasis, caused by Candida; and mucormycosis)), Pneumocystis carinii (pneumocystis pneumonia), atypical pneumonias (e.g., Mycoplasma and Chlamydia spp.), opportunistic infection pneumonia, nosocomial pneumonia, chemical pneumonitis, and aspiration pneumonia, pleural disorders (e.g., pleurisy, pleural effusion, and pneumothorax (e.g., simple spontaneous pneumothorax, complicated spontaneous pneumothorax, tension pneumothorax)), obstructive airway diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD), emphysema, chronic or acute bronchitis), occupational lung diseases (e.g., silicosis, black lung (coal workers' pneumoconiosis), asbestosis, berylliosis, occupational asthsma, byssinosis, and benign pneumoconioses), Infiltrative Lung Disease (e.g., pulmonary fibrosis (e.g., fibrosing alveolitis, usual interstitial pneumonia), idiopathic pulmonary fibrosis, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, histiocytosis X (e.g., Letterer-Siwe disease, Hand-Schüller-Christian disease, eosinophilic granuloma), idiopathic pulmonary hemosiderosis, sarcoidosis and pulmonary alveolar proteinosis), Acute respiratory distress syndrome (also called, e.g., adult respiratory distress syndrome), edema, pulmonary embolism, bronchitis (e.g., viral, bacterial), bronchiectasis, atelectasis, lung abscess (caused by, e.g., Staphylococcus aureus or Legionella pneumophila), and cystic fibrosis.

Anti Angiogenesis Activity

The naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis is one in which inhibitory influences predominate. Rastinejad et al., Cell 56:345-355 (1989). In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkman et al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985); Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science 221:719-725 (1983). In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data have accumulated which suggest that the growth of solid tumors is dependent on angiogenesis. Folkman and Klagsbrun, Science 235:442-447 (1987).

The present invention provides for treatment of diseases or disorders associated with neovascularization by administration of the polynucleotides and/or polypeptides of the invention, as well as agonists or antagonists of the present invention. Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)). Thus, the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the invention. For example, polynucleotides, polypeptides, antagonists and/or agonists may be utilized in a variety of additional methods in order to therapeutically treat a cancer or tumor. Cancers which may be treated with polynucleotides, polypeptides, antagonists and/or agonists include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lung cancer; colorectal cancer; advanced malignancies; and blood born tumors such as leukemias. For example, polynucleotides, polypeptides, antagonists and/or agonists may be delivered topically, in order to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.

Within yet other aspects, polynucleotides, polypeptides, antagonists and/or agonists may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration. Polynucleotides, polypeptides, antagonists and/or agonists may be delivered directly into the tumor, or near the tumor site, via injection or a catheter. Of course, as the artisan of ordinary skill will appreciate, the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and/or agonists may be useful in treating other disorders, besides cancers, which involve angiogenesis. These disorders include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis.

For example, within one aspect of the present invention methods are provided for treating hypertrophic scars and keloids, comprising the step of administering a polynucleotide, polypeptide, antagonist and/or agonist of the invention to a hypertrophic scar or keloid.

Within one embodiment of the present invention polynucleotides, polypeptides, antagonists and/or agonists of the invention are directly injected into a hypertrophic scar or keloid, in order to prevent the progression of these lesions. This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., burns), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development. As noted above, the present invention also provides methods for treating neovascular diseases of the eye, including for example, corneal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.

Moreover, Ocular disorders associated with neovascularization which can be treated with the polynucleotides and polypeptides of the present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312 (1978).

Thus, within one aspect of the present invention methods are provided for treating neovascular diseases of the eye such as corneal neovascularization (including corneal graft neovascularization), comprising the step of administering to a patient a therapeutically effective amount of a compound (as described above) to the cornea, such that the formation of blood vessels is inhibited. Briefly, the cornea is a tissue that normally lacks blood vessels. In certain pathological conditions however, capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus. When the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Visual loss may become complete if the cornea completely opacitates. A wide variety of disorders can result in corneal neovascularization, including for example, corneal infections (e.g., trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (e.g., graft rejection and Stevens-Johnson's syndrome), alkali burns, trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.

Particularly preferred embodiments of the invention may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form. The solution or suspension may be prepared in its pure form and administered several times daily. Alternatively, anti-angiogenic compositions, prepared as described above, may also be administered directly to the cornea. Within preferred embodiments, the anti-angiogenic composition is prepared with a muco-adhesive polymer that binds to cornea. Within further embodiments, the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy. Topical therapy may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.

Within other embodiments, the compounds described above may be injected directly into the corneal stroma by an ophthalmologist under microscopic guidance. The preferred site of injection may vary with the morphology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic corneal injection to “protect” the cornea from the advancing blood vessels. This method may also be utilized shortly after a corneal insult in order to prophylactically prevent corneal neovascularization. In this situation the material could be injected in the perilimbic cornea interspersed between the corneal lesion and its undesired potential limbic blood supply. Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections might only be required 2-3 times per year. A steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.

Within another aspect of the present invention, methods are provided for treating neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. In one embodiment, the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma. Within other embodiments, the compound may be implanted by injection into the region of the anterior chamber angle. Within other embodiments, the compound may also be placed in any location such that the compound is continuously released into the aqueous humor. Within another aspect of the present invention, methods are provided for treating proliferative diabetic retinopathy, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention, proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina. Preferably, this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.

Within another aspect of the present invention, methods are provided for treating retrolental fibroplasia, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eye, such that the formation of blood vessels is inhibited. The compound may be administered topically, via intravitreous injection and/or via intraocular implants.

Additionally, disorders which can be treated with the polynucleotides, polypeptides, agonists and/or agonists include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

Moreover, disorders and/or states, which can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated with the polynucleotides, polypeptides, agonists and/or agonists of the invention include, but are not limited to, solid tumors, blood born tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (keloids), nonunion fractures, scleroderma, trachoma, vascular adhesions, myocardial angiogenesis, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control agent by preventing vascularization required for embryo implantation controlling menstruation, diseases that have angiogenesis as a pathologic consequence such as cat scratch disease (Rochele minalia quintosa), ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a “morning after” method. Polynucleotides, polypeptides, agonists and/or agonists may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.

Polynucleotides, polypeptides, agonists and/or agonists of the present invention may be incorporated into surgical sutures in order to prevent stitch granulomas.

Polynucleotides, polypeptides, agonists and/or agonists may be utilized in a wide variety of surgical procedures. For example, within one aspect of the present invention a compositions (in the form of, for example, a spray or film) may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to surrounding tissues. Within other aspects of the present invention, compositions (e.g., in the form of a spray) may be delivered via endoscopic procedures in order to coat tumors, or inhibit angiogenesis in a desired locale. Within yet other aspects of the present invention, surgical meshes which have been coated with anti-angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized. For example, within one embodiment of the invention a surgical mesh laden with an anti-angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are provided for treating tumor excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist to the resection margins of a tumor subsequent to excision, such that the local recurrence of cancer and the formation of new blood vessels at the site is inhibited. Within one embodiment of the invention, the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound). Alternatively, the anti-angiogenic compounds may be incorporated into known surgical pastes prior to administration. Within particularly preferred embodiments of the invention, the anti-angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.

Within one aspect of the present invention, polynucleotides, polypeptides, agonists and/or agonists may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors. For example, within one embodiment of the invention, anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.

The polynucleotides, polypeptides, agonists and/or agonists of the present invention may also be administered along with other anti-angiogenic factors. Representative examples of other anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.

Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, 1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene (National Cancer Institute); Lobenzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; Takeuchi et al., Agents Actions 36:312-316, 1992); Thalidomide; Angiostatic steroid; AGM-1470; carboxynaminolmidazole; and metalloproteinase inhibitors such as BB94.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition of apoptosis that could be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated using polynucleotides or polypeptides, as well as antagonists or agonists of the present invention, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft v. host disease, acute graft rejection, and chronic graft rejection.

In preferred embodiments, polynucleotides, polypeptides, and/or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.

Additional diseases or conditions associated with increased cell survival that could be treated or detected by polynucleotides or polypeptides, or agonists or antagonists of the present invention include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis that could be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated using polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, include, but are not limited to, AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v. host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, for therapeutic purposes, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, and to stimulate hair follicle production and healing of dermal wounds. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associated with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to promote dermal reestablishment subsequent to dermal loss.

Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are types of grafts that polynucleotides or polypeptides, agonists or antagonists of the present invention, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hyperplastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, can be used to promote skin strength and to improve the appearance of aged skin.

It is believed that polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intestine, and large intestine. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract. Polynucleotides or polypeptides, agonists or antagonists of the present invention, may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may have a cytoprotective effect on the small intestine mucosa. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.

Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could further be used in full regeneration of skin in full and partial thickness skin defects, including burns, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly. Inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively. Thus, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease. Treatment with polynucleotides or polypeptides, agonists or antagonists of the present invention, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to treat diseases associate with the under expression.

Moreover, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to prevent and heal damage to the lungs due to various pathological states. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage. For example, emphysema, which results in the progressive loss of alveoli, and inhalation injuries, i.e., resulting from smoke inhalation and burns, that cause necrosis of the bronchiolar epithelium and alveoli could be effectively treated using polynucleotides or polypeptides, agonists or antagonists of the present invention. Also, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary dysplasia, in premature infants.

Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by cirrhosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).

In addition, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used treat or prevent the onset of diabetes mellitus. In patients with newly diagnosed Types I and II diabetes, where some islet cell function remains, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease. Also, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.

Neural Activity and Neurological Diseases

The polynucleotides, polypeptides and agonists or antagonists of the invention may be used for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases, disorders, damage or injury of the brain and/or nervous system. Nervous system disorders that can be treated with the compositions of the invention (e.g., polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the methods of the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to, degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including, but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

In one embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of hypoxia. In a further preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to protect neural cells from the damaging effects of cerebral hypoxia. According to this embodiment, the compositions of the invention are used to treat or prevent neural cell injury associated with cerebral hypoxia. In one non-exclusive aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention, are used to treat or prevent neural cell injury associated with cerebral ischemia. In another non-exclusive aspect of this embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with cerebral infarction.

In another preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with a stroke. In a specific embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent cerebral neural cell injury associated with a stroke.

In another preferred embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with a heart attack. In a specific embodiment, the polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent cerebral neural cell injury associated with a heart attack.

The compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in culture either in the presence or absence of hypoxia or hypoxic conditions; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in vivo. Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, in Zhang et al., Proc Natl Acad Sci USA 97:3637-42 (2000) or in Arakawa et al., J. Neurosci., 10:3507-15 (1990); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann. Rev. Neurosci., 4:17-42 (1981); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron disorders that may be treated according to the invention include, but are not limited to, disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Further, polypeptides or polynucleotides of the invention may play a role in neuronal survival; synapse formation; conductance; neural differentiation, etc. Thus, compositions of the invention (including polynucleotides, polypeptides, and agonists or antagonists) may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate diseases or disorders associated with these roles, including, but not limited to, learning and/or cognition disorders. The compositions of the invention may also be useful in the treatment or prevention of neurodegenerative disease states and/or behavioural disorders. Such neurodegenerative disease states and/or behavioral disorders include, but are not limited to, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease, Tourette Syndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder, panic disorder, learning disabilities, ALS, psychoses, autism, and altered behaviors, including disorders in feeding, sleep patterns, balance, and perception. In addition, compositions of the invention may also play a role in the treatment, prevention and/or detection of developmental disorders associated with the developing embryo, or sexually-linked disorders.

Additionally, polypeptides, polynucleotides and/or agonists or antagonists of the invention, may be useful in protecting neural cells from diseases, damage, disorders, or injury, associated with cerebrovascular disorders including, but not limited to, carotid artery diseases (e.g., carotid artery thrombosis, carotid stenosis, or Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis (e.g., carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome), cerebral hemorrhage (e.g., epidural or subdural hematoma, or subarachnoid hemorrhage), cerebral infarction, cerebral ischemia (e.g., transient cerebral ischemia, Subclavian Steal Syndrome, or vertebrobasilar insufficiency), vascular dementia (e.g., multi-infarct), leukomalacia, periventricular, and vascular headache (e.g., cluster headache or migraines).

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, for therapeutic purposes, for example, to stimulate neurological cell proliferation and/or differentiation. Therefore, polynucleotides, polypeptides, agonists and/or antagonists of the invention may be used to treat and/or detect neurologic diseases. Moreover, polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used as a marker or detector of a particular nervous system disease or disorder.

Examples of neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include brain diseases, such as metabolic brain diseases which includes phenylketonuria such as maternal phenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenase complex deficiency, Wernicke's Encephalopathy, brain edema, brain neoplasms such as cerebellar neoplasms which include infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavan disease, cerebellar diseases such as cerebellar ataxia which include spinocerebellar degeneration such as ataxia telangiectasia, cerebellar dyssynergia, Friederich's Ataxia, Machado-Joseph Disease, olivopontocerebellar atrophy, cerebellar neoplasms such as infratentorial neoplasms, diffuse cerebral sclerosis such as encephalitis periaxialis, globoid cell leukodystrophy, metachromatic leukodystrophy and subacute sclerosing panencephalitis.

Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include cerebrovascular disorders (such as carotid artery diseases which include carotid artery thrombosis, carotid stenosis and Moyamoya Disease), cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations, cerebral artery diseases, cerebral embolism and thrombosis such as carotid artery thrombosis, sinus thrombosis and Wallenberg's Syndrome, cerebral hemorrhage such as epidural hematoma, subdural hematoma and subarachnoid hemorrhage, cerebral infarction, cerebral ischemia such as transient cerebral ischemia, Subclavian Steal Syndrome and vertebrobasilar insufficiency, vascular dementia such as multi-infarct dementia, periventricular leukomalacia, vascular headache such as cluster headache and migraine.

Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include dementia such as AIDS Dementia Complex, presenile dementia such as Alzheimer's Disease and Creutzfeldt-Jakob Syndrome, senile dementia such as Alzheimer's Disease and progressive supranuclear palsy, vascular dementia such as multi-infarct dementia, encephalitis which include encephalitis periaxialis, viral encephalitis such as epidemic encephalitis, Japanese Encephalitis, St. Louis Encephalitis, tick-borne encephalitis and West Nile Fever, acute disseminated encephalomyelitis, meningoencephalitis such as uveomeningoencephalitic syndrome, Postencephalitic Parkinson Disease and subacute sclerosing panencephalitis, encephalomalacia such as periventricular leukomalacia, epilepsy such as generalized epilepsy which includes infantile spasms, absence epilepsy, myoclonic epilepsy which includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsy such as complex partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic epilepsy, status epilepticus such as Epilepsia Partialis Continua, and Hallervorden-Spatz Syndrome.

Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include hydrocephalus such as Dandy-Walker Syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, narcolepsy which includes cataplexy, bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome, Reye's Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranial tuberculoma and Zellweger Syndrome, central nervous system infections such as AIDS Dementia Complex, Brain Abscess, subdural empyema, encephalomyelitis such as Equine Encephalomyelitis, Venezuelan Equine Encephalomyelitis, Necrotizing Hemorrhagic Encephalomyelitis, Visna, and cerebral malaria.

Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include meningitis such as arachnoiditis, aseptic meningtitis such as viral meningtitis which includes lymphocytic choriomeningitis, Bacterial meningtitis which includes Haemophilus Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such as Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal tuberculosis, fungal meningitis such as Cryptococcal Meningtitis, subdural effusion, meningoencephalitis such as uvemeningoencephalitic syndrome, myelitis such as transverse myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which includes bulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases (such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie), and cerebral toxoplasmosis.

Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include central nervous system neoplasms such as brain neoplasms that include cerebellar neoplasms such as infratentorial neoplasms, cerebral ventricle neoplasms such as choroid plexus neoplasms, hypothalamic neoplasms and supratentorial neoplasms, meningeal neoplasms, spinal cord neoplasms which include epidural neoplasms, demyelinating diseases such as Canavan Diseases, diffuse cerebral sceloris which includes adrenoleukodystrophy, encephalitis periaxialis, globoid cell leukodystrophy, diffuse cerebral sclerosis such as metachromatic leukodystrophy, allergic encephalomyelitis, necrotizing hemorrhagic encephalomyelitis, progressive multifocal leukoencephalopathy, multiple sclerosis, central pontine myelinolysis, transverse myelitis, neuromyelitis optica, Scrapie, Swayback, Chronic Fatigue Syndrome, Visna, High Pressure Nervous Syndrome, Meningism, spinal cord diseases such as amyotonia congenita, amyotrophic lateral sclerosis, spinal muscular atrophy such as Werdnig-Hoffmann Disease, spinal cord compression, spinal cord neoplasms such as epidural neoplasms, syringomyelia, Tabes Dorsalis, Stiff-Man Syndrome, mental retardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange's Syndrome, Down Syndrome, Gangliosidoses such as gangliosidoses G(M1), Sandhoff Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria, Laurence-Moon-Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup Urine Disease, mucolipidosis such as fucosidosis, neuronal ceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria such as maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome, Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome, nervous system abnormalities such as holoprosencephaly, neural tube defects such as anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity, encephalocele, meningocele, meningomyelocele, spinal dysraphism such as spina bifida cystica and spina bifida occulta.

Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include hereditary motor and sensory neuropathies which include Charcot-Marie Disease, Hereditary optic atrophy, Refsum's Disease, hereditary spastic paraplegia, Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies such as Congenital Analgesia and Familial Dysautonomia, Neurologic manifestations (such as agnosia that include Gerstmann's Syndrome, Amnesia such as retrograde amnesia, apraxia, neurogenic bladder, cataplexy, communicative disorders such as hearing disorders that includes deafness, partial hearing loss, loudness recruitment and tinnitus, language disorders such as aphasia which include agraphia, anomia, broca aphasia, and Wernicke Aphasia, Dyslexia such as Acquired Dyslexia, language development disorders, speech disorders such as aphasia which includes anomia, broca aphasia and Wernicke Aphasia, articulation disorders, communicative disorders such as speech disorders which include dysarthria, echolalia, mutism and stuttering, voice disorders such as aphonia and hoarseness, decerebrate state, delirium, fasciculation, hallucinations, meningism, movement disorders such as angelman syndrome, ataxia, athetosis, chorea, dystonia, hypokinesia, muscle hypotonia, myoclonus, tic, torticollis and tremor, muscle hypertonia such as muscle rigidity such as stiff-man syndrome, muscle spasticity, paralysis such as facial paralysis which includes Herpes Zoster Oticus, Gastroparesis, Hemiplegia, ophthalmoplegia such as diplopia, Duane's Syndrome, Horner's Syndrome, Chronic progressive external ophthalmoplegia such as Kearns Syndrome, Bulbar Paralysis, Tropical Spastic Paraparesis, Paraplegia such as Brown-Sequard Syndrome, quadriplegia, respiratory paralysis and vocal cord paralysis, paresis, phantom limb, taste disorders such as ageusia and dysgeusia, vision disorders such as amblyopia, blindness, color vision defects, diplopia, hemianopsia, scotoma and subnormal vision, sleep disorders such as hypersomnia which includes Kleine-Levin Syndrome, insomnia, and somnambulism, spasm such as trismus, unconsciousness such as coma, persistent vegetative state and syncope and vertigo, neuromuscular diseases such as amyotonia congenita, amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motor neuron disease, muscular atrophy such as spinal muscular atrophy, Charcot-Marie Disease and Werdnig-Hoffmann Disease, Postpoliomyelitis Syndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia Atrophica, Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome, peripheral nervous system diseases such as acrodynia, amyloid neuropathies, autonomic nervous system diseases such as Adie's Syndrome, Barre-Lieou Syndrome, Familial Dysautonomia, Horner's Syndrome, Reflex Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseases such as Acoustic Nerve Diseases such as Acoustic Neuroma which includes Neurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia, Melkersson-Rosenthal Syndrome, ocular motility disorders which includes amblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia such as Duane's Syndrome, Horner's Syndrome, Chronic Progressive External Ophthalmoplegia which includes Kearns Syndrome, Strabismus such as Esotropia and Exotropia, Oculomotor Nerve Paralysis, Optic Nerve Diseases such as Optic Atrophy which includes Hereditary Optic Atrophy, Optic Disk Drusen, Optic Neuritis such as Neuromyelitis Optica, Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis, Demyelinating Diseases such as Neuromyelitis Optica and Swayback, and Diabetic neuropathies such as diabetic foot.

Additional neurologic diseases which can be treated or detected with polynucleotides, polypeptides, agonists, and/or antagonists of the present invention include nerve compression syndromes such as carpal tunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome such as cervical rib syndrome, ulnar nerve compression syndrome, neuralgia such as causalgia, cervico-brachial neuralgia, facial neuralgia and trigeminal neuralgia, neuritis such as experimental allergic neuritis, optic neuritis, polyneuritis, polyradiculoneuritis and radiculities such as polyradiculitis, hereditary motor and sensory neuropathies such as Charcot-Marie Disease, Hereditary Optic Atrophy, Refsum's Disease, Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, Hereditary Sensory and Autonomic Neuropathies which include Congenital Analgesia and Familial Dysautonomia, POEMS Syndrome, Sciatica, Gustatory Sweating and Tetany).

Endocrine Disorders

Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate disorders and/or diseases related to hormone imbalance, and/or disorders or diseases of the endocrine system.

Hormones secreted by the glands of the endocrine system control physical growth, sexual function, metabolism, and other functions. Disorders may be classified in two ways: disturbances in the production of hormones, and the inability of tissues to respond to hormones. The etiology of these hormone imbalance or endocrine system diseases, disorders or conditions may be genetic, somatic, such as cancer and some autoimmune diseases, acquired (e.g., by chemotherapy, injury or toxins), or infectious. Moreover, polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention can be used as a marker or detector of a particular disease or disorder related to the endocrine system and/or hormone imbalance.

Endocrine system and/or hormone imbalance and/or diseases encompass disorders of uterine motility including, but not limited to: complications with pregnancy and labor (e.g., pre-term labor, post-term pregnancy, spontaneous abortion, and slow or stopped labor); and disorders and/or diseases of the menstrual cycle (e.g., dysmenorrhea and endometriosis).

Endocrine system and/or hormone imbalance disorders and/or diseases include disorders and/or diseases of the pancreas, such as, for example, diabetes mellitus, diabetes insipidus, congenital pancreatic agenesis, pheochromocytoma—islet cell tumor syndrome; disorders and/or diseases of the adrenal glands such as, for example, Addison's Disease, corticosteroid deficiency, virilizing disease, hirsutism, Cushing's Syndrome, hyperaldosteronism, pheochromocytoma; disorders and/or diseases of the pituitary gland, such as, for example, hyperpituitarism, hypopituitarism, pituitary dwarfism, pituitary adenoma, panhypopituitarism, acromegaly, gigantism; disorders and/or diseases of the thyroid, including but not limited to, hyperthyroidism, hypothyroidism, Plummer's disease, Graves' disease (toxic diffuse goiter), toxic nodular goiter, thyroiditis (Hashimoto's thyroiditis, subacute granulomatous thyroiditis, and silent lymphocytic thyroiditis), Pendred's syndrome, myxedema, cretinism, thyrotoxicosis, thyroid hormone coupling defect, thymic aplasia, Hurthle cell tumours of the thyroid, thyroid cancer, thyroid carcinoma, Medullary thyroid carcinoma; disorders and/or diseases of the parathyroid, such as, for example, hyperparathyroidism, hypoparathyroidism; disorders and/or diseases of the hypothalamus.

In specific embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists of those polypeptides (including antibodies) as well as fragments and variants of those polynucleotides, polypeptides, agonists and antagonists, may be used to diagnose, prognose, treat, prevent, or ameliorate diseases and disorders associated with aberrant glucose metabolism or glucose uptake into cells.

In a specific embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists and/or antagonists thereof may be used to diagnose, prognose, treat, prevent, and/or ameliorate type I diabetes mellitus (insulin dependent diabetes mellitus, IDDM).

In another embodiment, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists and/or antagonists thereof may be used to diagnose, prognose, treat, prevent, and/or ameliorate type II diabetes mellitus (insulin resistant diabetes mellitus).

Additionally, in other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or antagonists thereof (especially neutralizing or antagonistic antibodies) may be used to diagnose, prognose, treat, prevent, and/or ameliorate conditions associated with (type I or type II) diabetes mellitus, including, but not limited to, diabetic ketoacidosis, diabetic coma, nonketotic hyperglycemic-hyperosmolar coma, seizures, mental confusion, drowsiness, cardiovascular disease (e.g., heart disease, atherosclerosis, microvascular disease, hypertension, stroke, and other diseases and disorders as described in the “Cardiovascular Disorders” section), dyslipidemia, kidney disease (e.g., renal failure, nephropathy other diseases and disorders as described in the “Renal Disorders” section), nerve damage, neuropathy, vision impairment (e.g., diabetic retinopathy and blindness), ulcers and impaired wound healing, infections (e.g., infectious diseases and disorders as described in the “Infectious Diseases” section, especially of the urinary tract and skin), carpal tunnel syndrome and Dupuytren's contracture.

In other embodiments, the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to regulate the animal's weight. In specific embodiments the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to control the animal's weight by modulating a biochemical pathway involving insulin. In still other embodiments the polynucleotides and/or polypeptides corresponding to this gene and/or agonists or antagonists thereof are administered to an animal, preferably a mammal, and most preferably a human, in order to control the animal's weight by modulating a biochemical pathway involving insulin-like growth factor.

In addition, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases of the testes or ovaries, including cancer. Other disorders and/or diseases of the testes or ovaries further include, for example, ovarian cancer, polycystic ovary syndrome, Klinefelter's syndrome, vanishing testes syndrome (bilateral anorchia), congenital absence of Leydig's cells, cryptorchidism, Noonan's syndrome, myotonic dystrophy, capillary haemangioma of the testis (benign), neoplasias of the testis and neo-testis.

Moreover, endocrine system and/or hormone imbalance disorders and/or diseases may also include disorders and/or diseases such as, for example, polyglandular deficiency syndromes, pheochromocytoma, neuroblastoma, multiple Endocrine neoplasia, and disorders and/or cancers of endocrine tissues.

In another embodiment, a polypeptide of the invention, or polynucleotides, antibodies, agonists, or antagonists corresponding to that polypeptide, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate endocrine diseases and/or disorders associated with the tissue(s) in which the polypeptide of the invention is expressed, including one, two, three, four, five, or more tissues disclosed in Table 1B.2, column 5 (Tissue Distribution Library Code).

Reproductive System Disorders

The polynucleotides or polypeptides, or agonists or antagonists of the invention may be used for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases and/or disorders of the reproductive system. Reproductive system disorders that can be treated by the compositions of the invention, include, but are not limited to, reproductive system injuries, infections, neoplastic disorders, congenital defects, and diseases or disorders which result in infertility, complications with pregnancy, labor, or parturition, and postpartum difficulties.

Reproductive system disorders and/or diseases include diseases and/or disorders of the testes, including testicular atrophy, testicular feminization, cryptorchism (unilateral and bilateral), anorchia, ectopic testis, epididymitis and orchitis (typically resulting from infections such as, for example, gonorrhea, mumps, tuberculosis, and syphilis), testicular torsion, vasitis nodosa, germ cell tumors (e.g., seminomas, embryonal cell carcinomas, teratocarcinomas, choriocarcinomas, yolk sac tumors, and teratomas), stromal tumors (e.g., Leydig cell tumors), hydrocele, hematocele, varicocele, spermatocele, inguinal hernia, and disorders of sperm production (e.g., immotile cilia syndrome, aspermia, asthenozoospermia, azoospermia, oligospermia, and teratozoospermia).

Reproductive system disorders also include disorders of the prostate gland, such as acute non-bacterial prostatitis, chronic non-bacterial prostatitis, acute bacterial prostatitis, chronic bacterial prostatitis, prostatodystonia, prostatosis, granulomatous prostatitis, malacoplakia, benign prostatic hypertrophy or hyperplasia, and prostate neoplastic disorders, including adenocarcinomas, transitional cell carcinomas, ductal carcinomas, and squamous cell carcinomas.

Additionally, the compositions of the invention may be useful in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of disorders or diseases of the penis and urethra, including inflammatory disorders, such as balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, syphilis, herpes simplex virus, gonorrhea, non-gonococcal urethritis, chlamydia, mycoplasma, trichomonas, HIV, AIDS, Reiter's syndrome, condyloma acuminatum, condyloma latum, and pearly penile papules; urethral abnormalities, such as hypospadias, epispadias, and phimosis; premalignant lesions, including Erythroplasia of Queyrat, Bowen's disease, Bowenoid paplosis, giant condyloma of Buscke-Lowenstein, and varrucous carcinoma; penile cancers, including squamous cell carcinomas, carcinoma in situ, verrucous carcinoma, and disseminated penile carcinoma; urethral neoplastic disorders, including penile urethral carcinoma, bulbomembranous urethral carcinoma, and prostatic urethral carcinoma; and erectile disorders, such as priapism, Peyronie's disease, erectile dysfunction, and impotence.

Moreover, diseases and/or disorders of the vas deferens include vasculititis and CBAVD (congenital bilateral absence of the vas deferens); additionally, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases and disorders of the seminal vesicles, including hydatid disease, congenital chloride diarrhea, and polycystic kidney disease.

Other disorders and/or diseases of the male reproductive system include, for example, Klinefelter's syndrome, Young's syndrome, premature ejaculation, diabetes mellitus, cystic fibrosis, Kartagener's syndrome, high fever, multiple sclerosis, and gynecomastia.

Further, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases and/or disorders of the vagina and vulva, including bacterial vaginosis, candida vaginitis, herpes simplex virus, chancroid, granuloma inguinale, lymphogranuloma venereum, scabies, human papillomavirus, vaginal trauma, vulvar trauma, adenosis, chlamydia vaginitis, gonorrhea, trichomonas vaginitis, condyloma acuminatum, syphilis, molluscum contagiosum, atrophic vaginitis, Paget's disease, lichen sclerosus, lichen planus, vulvodynia, toxic shock syndrome, vaginismus, vulvovaginitis, vulvar vestibulitis, and neoplastic disorders, such as squamous cell hyperplasia, clear cell carcinoma, basal cell carcinoma, melanomas, cancer of Bartholin's gland, and vulvar intraepithelial neoplasia.

Disorders and/or diseases of the uterus include dysmenorrhea, retroverted uterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding, amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome, premature menopause, precocious puberty, uterine polyps, dysfunctional uterine bleeding (e.g., due to aberrant hormonal signals), and neoplastic disorders, such as adenocarcinomas, keiomyosarcomas, and sarcomas. Additionally, the polypeptides, polynucleotides, or agonists or antagonists of the invention may be useful as a marker or detector of, as well as in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of congenital uterine abnormalities, such as bicornuate uterus, septate uterus, simple unicornuate uterus, unicornuate uterus with a noncavitary rudimentary horn, unicornuate uterus with a non-communicating cavitary rudimentary horn, unicornuate uterus with a communicating cavitary horn, arcuate uterus, uterine didelfus, and T-shaped uterus.

Ovarian diseases and/or disorders include anovulation, polycystic ovary syndrome (Stein-Leventhal syndrome), ovarian cysts, ovarian hypofunction, ovarian insensitivity to gonadotropins, ovarian overproduction of androgens, right ovarian vein syndrome, amenorrhea, hirutism, and ovarian cancer (including, but not limited to, primary and secondary cancerous growth, Sertoli-Leydig tumors, endometriod carcinoma of the ovary, ovarian papillary serous adenocarcinoma, ovarian mucinous adenocarcinoma, and Ovarian Krukenberg tumors).

Cervical diseases and/or disorders include cervicitis, chronic cervicitis, mucopurulent cervicitis, cervical dysplasia, cervical polyps, Nabothian cysts, cervical erosion, cervical incompetence, and cervical neoplasms (including, for example, cervical carcinoma, squamous metaplasia, squamous cell carcinoma, adenosquamous cell neoplasia, and columnar cell neoplasia).

Additionally, diseases and/or disorders of the reproductive system include disorders and/or diseases of pregnancy, including miscarriage and stillbirth, such as early abortion, late abortion, spontaneous abortion, induced abortion, therapeutic abortion, threatened abortion, missed abortion, incomplete abortion, complete abortion, habitual abortion, missed abortion, and septic abortion; ectopic pregnancy, anemia, Rh incompatibility, vaginal bleeding during pregnancy, gestational diabetes, intrauterine growth retardation, polyhydramnios, HELLP syndrome, abruptio placentae, placenta previa, hyperemesis, preeclampsia, eclampsia, herpes gestationis, and urticaria of pregnancy. Additionally, the polynucleotides, polypeptides, and agonists or antagonists of the present invention may be used in the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of diseases that can complicate pregnancy, including heart disease, heart failure, rheumatic heart disease, congenital heart disease, mitral valve prolapse, high blood pressure, anemia, kidney disease, infectious disease (e.g., rubella, cytomegalovirus, toxoplasmosis, infectious hepatitis, chlamydia, HIV, AIDS, and genital herpes), diabetes mellitus, Graves' disease, thyroiditis, hypothyroidism, Hashimoto's thyroiditis, chronic active hepatitis, cirrhosis of the liver, primary biliary cirrhosis, asthma, systemic lupus eryematosis, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, appendicitis, ovarian cysts, gallbladder disorders, and obstruction of the intestine.

Complications associated with labor and parturition include premature rupture of the membranes, pre-term labor, post-term pregnancy, postmaturity, labor that progresses too slowly, fetal distress (e.g., abnormal heart rate (fetal or maternal), breathing problems, and abnormal fetal position), shoulder dystocia, prolapsed umbilical cord, amniotic fluid embolism, and aberrant uterine bleeding.

Further, diseases and/or disorders of the postdelivery period, including endometritis, myometritis, parametritis, peritonitis, pelvic thrombophlebitis, pulmonary embolism, endotoxemia, pyelonephritis, saphenous thrombophlebitis, mastitis, cystitis, postpartum hemorrhage, and inverted uterus.

Other disorders and/or diseases of the female reproductive system that may be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by the polynucleotides, polypeptides, and agonists or antagonists of the present invention include, for example, Turner's syndrome, pseudohermaphroditism, premenstrual syndrome, pelvic inflammatory disease, pelvic congestion (vascular engorgement), frigidity, anorgasmia, dyspareunia, ruptured fallopian tube, and Mittelschmerz.

Infectious Disease

Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention. Examples of viruses, include, but are not limited to Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat AIDS.

Similarly, bacterial and fungal agents that can cause disease or symptoms and that can be treated or detected by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following Gram-Negative and Gram-positive bacteria, bacterial families, and fungi: Actinomyces (e.g., Norcardia), Acinetobacter, Cryptococcus neoformans, Aspergillus, Bacillaceae (e.g., Bacillus anthrasis), Bacteroides (e.g., Bacteroides fragilis), Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi), Brucella, Candidia, Campylobacter, Chlamydia, Clostridium (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Coccidioides, Corynebacterium (e.g., Corynebacterium diptheriae), Cryptococcus, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacter (e.g. Enterobacter aerogenes), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, Salmonella enteritidis, Salmonella typhi), Serratia, Yersinia, Shigella), Erysipelothrix, Haemophilus (e.g., Haemophilus influenza type B), Helicobacter, Legionella (e.g., Legionella pneumophila), Leptospira, Listeria (e.g., Listeria monocytogenes), Mycoplasma, Mycobacterium (e.g., Mycobacterium leprae and Mycobacterium tuberculosis), Vibrio (e.g., Vibrio cholerae), Neisseriaceae (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pasteurellacea, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B), Pasteurella), Chlamydiaceae, Syphilis, Proteus, Pseudomonas (e.g., Pseudomonas aeruginosa), Rickettsiaceae, Spirochetes (e.g., Treponema spp., Leptospira spp., Borrelia spp.), Shigella spp., Staphylococcus (e.g., Staphylococcus aureus), Meningiococcus, Pneumococcus and Streptococcus (e.g., Streptococcus pneumoniae and Groups A, B, and C Streptococci), and Ureaplasmas. These bacterial, parasitic, and fungal families can cause diseases or symptoms, including, but not limited to: antibiotic-resistant infections, bacteremia, endocarditis, septicemia, eye infections (e.g., conjunctivitis), uveitis, tuberculosis, gingivitis, bacterial diarrhea, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, dental caries, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, dysentery, paratyphoid fever, food poisoning, Legionella disease, chronic and acute inflammation, erythema, yeast infections, typhoid, pneumonia, gonorrhea, meningitis (e.g., mengitis types A and B), chlamydia, syphillis, diphtheria, leprosy, brucellosis, peptic ulcers, anthrax, spontaneous abortions, birth defects, pneumonia, lung infections, ear infections, deafness, blindness, lethargy, malaise, vomiting, chronic diarrhea, Crohn's disease, colitis, vaginosis, sterility, pelvic inflammatory diseases, candidiasis, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections, noscomial infections. Polynucleotides or polypeptides, agonists or antagonists of the invention, can be used to treat or detect any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat: tetanus, diptheria, botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can be detected, prevented, diagnosed, prognosticated, treated, and/or ameliorated by a polynucleotide or polypeptide and/or agonist or antagonist of the present invention include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardias, Helminthiasis, Leishmaniasis, Schistisoma, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. polynucleotides or polypeptides, or agonists or antagonists of the invention, can be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate any of these symptoms or diseases. In specific embodiments, polynucleotides, polypeptides, or agonists or antagonists of the invention are used to detect, prevent, diagnose, treat, and/or ameliorate malaria.

Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy). Moreover, the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.

Regeneration

Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues. (See, Science 276:59-87 (1997)). The regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vasculature (including vascular and lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue. Preferably, regeneration occurs without or decreased scarring. Regeneration also may include angiogenesis.

Moreover, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage. Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects. A further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using polynucleotides or polypeptides, as well as agonists or antagonists of the present invention, to proliferate and differentiate nerve cells. Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke). Specifically, diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), could all be treated using the polynucleotides or polypeptides, as well as agonists or antagonists of the present invention.

Gastrointestinal Disorders

Polynucleotides or polypeptides, or agonists or antagonists of the present invention, may be used to detect, prevent, diagnose, prognosticate, treat, and/or ameliorate gastrointestinal diseases and disorders, including inflammatory diseases and/or conditions, infections, cancers (e.g., intestinal neoplasms (carcinoid tumor of the small intestine, non-Hodgkin's lymphoma of the small intestine, small bowl lymphoma)), and ulcers, such as peptic ulcers.

Gastrointestinal disorders include dysphagia, odynophagia, inflammation of the esophagus, peptic esophagitis, gastric reflux, submucosal fibrosis and stricturing, Mallory-Weiss lesions, leiomyomas, lipomas, epidermal cancers, adeoncarcinomas, gastric retention disorders, gastroenteritis, gastric atrophy, gastric/stomach cancers, polyps of the stomach, autoimmune disorders such as pernicious anemia, pyloric stenosis, gastritis (bacterial, viral, eosinophilic, stress-induced, chronic erosive, atrophic, plasma cell, and Ménétrier's), and peritoneal diseases (e.g., chyloperioneum, hemoperitoneum, mesenteric cyst, mesenteric lymphadenitis, mesenteric vascular occlusion, panniculitis, neoplasms, peritonitis, pneumoperitoneum, bubphrenic abscess).

Gastrointestinal disorders also include disorders associated with the small intestine, such as malabsorption syndromes, distension, irritable bowel syndrome, sugar intolerance, celiac disease, duodenal ulcers, duodenitis, tropical sprue, Whipple's disease, intestinal lymphangiectasia, Crohn's disease, appendicitis, obstructions of the ileum, Meckel's diverticulum, multiple diverticula, failure of complete rotation of the small and large intestine, lymphoma, and bacterial and parasitic diseases (such as Traveler's diarrhea, typhoid and paratyphoid, cholera, infection by Roundworms (Ascariasis lumbricoides), Hookworms (Ancylostoma duodenale), Threadworms (Enterobius vermicularis), Tapeworms (Taenia saginata, Echinococcus granulosus, Diphyllobothrium spp., and T. solium).

Liver diseases and/or disorders include intrahepatic cholestasis (alagille syndrome, biliary liver cirrhosis), fatty liver (alcoholic fatty liver, reye syndrome), hepatic vein thrombosis, hepatolentricular degeneration, hepatomegaly, hepatopulmonary syndrome, hepatorenal syndrome, portal hypertension (esophageal and gastric varices), liver abscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary and experimental), alcoholic liver diseases (fatty liver, hepatitis, cirrhosis), parasitic (hepatic echinococcosis, fascioliasis, amebic liver abscess), jaundice (hemolytic, hepatocellular, and cholestatic), cholestasis, portal hypertension, liver enlargement, ascites, hepatitis (alcoholic hepatitis, animal hepatitis, chronic hepatitis (autoimmune, hepatitis B, hepatitis C, hepatitis D, drug induced), toxic hepatitis, viral human hepatitis (hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E), Wilson's disease, granulomatous hepatitis, secondary biliary cirrhosis, hepatic encephalopathy, portal hypertension, varices, hepatic encephalopathy, primary biliary cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma, hemangiomas, bile stones, liver failure (hepatic encephalopathy, acute liver failure), and liver neoplasms (angiomyolipoma, calcified liver metastases, cystic liver metastases, epithelial tumors, fibrolamellar hepatocarcinoma, focal nodular hyperplasia, hepatic adenoma, hepatobiliary cystadenoma, hepatoblastoma, hepatocellular carcinoma, hepatoma, liver cancer, liver hemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors of liver, nodular regenerative hyperplasia, benign liver tumors (Hepatic cysts [Simple cysts, Polycystic liver disease, Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal tumors [Mesenchymal hamartoma, Infantile hemangioendothelioma, Hemangioma, Peliosis hepatis, Lipomas, Inflammatory pseudotumor, Miscellaneous], Epithelial tumors [Bile duct epithelium (Bile duct hamartoma, Bile duct adenoma), Hepatocyte (Adenoma, Focal nodular hyperplasia, Nodular regenerative hyperplasia)], malignant liver tumors [hepatocellular, hepatoblastoma, hepatocellular carcinoma, cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors of blood vessels, angiosarcoma, Karposi's sarcoma, hemangioendothelioma, other tumors, embryonal sarcoma, fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma, teratoma, carcinoid, squamous carcinoma, primary lymphoma]), peliosis hepatis, erythrohepatic porphyria, hepatic porphyria (acute intermittent porphyria, porphyria cutanea tarda), Zellweger syndrome).

Pancreatic diseases and/or disorders include acute pancreatitis, chronic pancreatitis (acute necrotizing pancreatitis, alcoholic pancreatitis), neoplasms (adenocarcinoma of the pancreas, cystadenocarcinoma, insulinoma, gastrinoma, and glucagonoma, cystic neoplasms, islet-cell tumors, pancreoblastoma), and other pancreatic diseases (e.g., cystic fibrosis, cyst (pancreatic pseudocyst, pancreatic fistula, insufficiency)).

Gallbladder diseases include gallstones (cholelithiasis and choledocholithiasis), postcholecystectomy syndrome, diverticulosis of the gallbladder, acute cholecystitis, chronic cholecystitis, bile duct tumors, and mucocele.

Diseases and/or disorders of the large intestine include antibiotic-associated colitis, diverticulitis, ulcerative colitis, acquired megacolon, abscesses, fungal and bacterial infections, anorectal disorders (e.g., fissures, hemorrhoids), colonic diseases (colitis, colonic neoplasms [colon cancer, adenomatous colon polyps (e.g., villous adenoma), colon carcinoma, colorectal cancer], colonic diverticulitis, colonic diverticulosis, megacolon [Hirschsprung disease, toxic megacolon]; sigmoid diseases [proctocolitis, sigmoin neoplasms]), constipation, Crohn's disease, diarrhea (infantile diarrhea, dysentery), duodenal diseases (duodenal neoplasms, duodenal obstruction, duodenal ulcer, duodenitis), enteritis (enterocolitis), HIV enteropathy, ileal diseases (ileal neoplasms, ileitis), immunoproliferative small intestinal disease, inflammatory bowel disease (ulcerative colitis, Crohn's disease), intestinal atresia, parasitic diseases (anisakiasis, balantidiasis, blastocystis infections, cryptosporidiosis, dientamoebiasis, amebic dysentery, giardiasis), intestinal fistula (rectal fistula), intestinal neoplasms (cecal neoplasms, colonic neoplasms, duodenal neoplasms, ileal neoplasms, intestinal polyps, jejunal neoplasms, rectal neoplasms), intestinal obstruction (afferent loop syndrome, duodenal obstruction, impacted feces, intestinal pseudo-obstruction [cecal volvulus], intussusception), intestinal perforation, intestinal polyps (colonic polyps, gardner syndrome, Peutz-jeghers syndrome), jejunal diseases (jejunal neoplasms), malabsorption syndromes (blind loop syndrome, celiac disease, lactose intolerance, short bowl syndrome, tropical sprue, whipple's disease), mesenteric vascular occlusion, pneumatosis cystoides intestinalis, protein-losing enteropathies (intestinal lymphagiectasis), rectal diseases (anus diseases, fecal incontinence, hemorrhoids, proctitis, rectal fistula, rectal prolapse, rectocele), peptic ulcer (duodenal ulcer, peptic esophagitis, hemorrhage, perforation, stomach ulcer, Zollinger-Ellison syndrome), postgastrectomy syndromes (dumping syndrome), stomach diseases (e.g., achlorhydria, duodenogastric reflux (bile reflux), gastric antral vascular ectasia, gastric fistula, gastric outlet obstruction, gastritis (atrophic or hypertrophic), gastroparesis, stomach dilatation, stomach diverticulum, stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, hyperplastic gastric polyp), stomach rupture, stomach ulcer, stomach volvulus), tuberculosis, visceroptosis, vomiting (e.g., hematemesis, hyperemesis gravidarum, postoperative nausea and vomiting) and hemorrhagic colitis.

Further diseases and/or disorders of the gastrointestinal system include biliary tract diseases, such as, gastroschisis, fistula (e.g., biliary fistula, esophageal fistula, gastric fistula, intestinal fistula, pancreatic fistula), neoplasms (e.g., biliary tract neoplasms, esophageal neoplasms, such as adenocarcinoma of the esophagus, esophageal squamous cell carcinoma, gastrointestinal neoplasms, pancreatic neoplasms, such as adenocarcinoma of the pancreas, mucinous cystic neoplasm of the pancreas, pancreatic cystic neoplasms, pancreatoblastoma, and peritoneal neoplasms), esophageal disease (e.g., bullous diseases, candidiasis, glycogenic acanthosis, ulceration, barrett esophagus varices, atresia, cyst, diverticulum (e.g., Zenker's diverticulum), fistula (e.g., tracheoesophageal fistula), motility disorders (e.g., CREST syndrome, deglutition disorders, achalasia, spasm, gastroesophageal reflux), neoplasms, perforation (e.g., Boerhaave syndrome, Mallory-Weiss syndrome), stenosis, esophagitis, diaphragmatic hernia (e.g., hiatal hernia); gastrointestinal diseases, such as, gastroenteritis (e.g., cholera morbus, norwalk virus infection), hemorrhage (e.g., hematemesis, melena, peptic ulcer hemorrhage), stomach neoplasms (gastric cancer, gastric polyps, gastric adenocarcinoma, stomach cancer)), hernia (e.g., congenital diaphragmatic hernia, femoral hernia, inguinal hernia, obturator hernia, umbilical hernia, ventral hernia), and intestinal diseases (e.g., cecal diseases (appendicitis, cecal neoplasms)).

Chemotaxis

Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may have chemotaxis activity. A chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation. The mobilized cells can then fight off and/or heal the particular trauma or abnormality.

Polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.

It is also contemplated that polynucleotides or polypeptides, as well as agonists or antagonists of the present invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, polynucleotides or polypeptides, as well as agonists or antagonists of the present invention could be used as an inhibitor of chemotaxis.

Binding Activity

A polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic. (See, Coligan et al., Current Protocols in Immunology 1(2):Chapter 5 (1991)). Similarly, the molecule can be closely related to the natural receptor to which the polypeptide binds, or at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., active site). In either case, the molecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producing appropriate cells which express the polypeptide. Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.

Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody. The antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.

Additionally, the receptor to which the polypeptide of the present invention binds can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to the polypeptides, for example, NIH3T3 cells which are known to contain multiple receptors for the FGF family proteins, and SC-3 cells, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to the polypeptides. Transfected cells which are grown on glass slides are exposed to the polypeptide of the present invention, after they have been labeled. The polypeptides can be labeled by a variety of means including iodination or inclusion of a recognition site for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clones that encodes the putative receptor.

As an alternative approach for receptor identification, the labeled polypeptides can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptors of the polypeptides can be excised, resolved into peptide fragments, and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the genes encoding the putative receptors.

Moreover, the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) may be employed to modulate the activities of the polypeptide of the present invention thereby effectively generating agonists and antagonists of the polypeptide of the present invention. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques 24(2):308-13 (1998); each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of polynucleotides and corresponding polypeptides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired molecule by homologous, or site-specific, recombination. In another embodiment, polynucleotides and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of the polypeptide of the present invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are family members. In further preferred embodiments, the heterologous molecule is a growth factor such as, for example, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, and glial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of the polypeptide of the present invention. Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention. The biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.

Additionally, this invention provides a method of screening compounds to identify those which modulate the action of the polypeptide of the present invention. An example of such an assay comprises combining a mammalian fibroblast cell, a the polypeptide of the present invention, the compound to be screened and ³[H] thymidine under cell culture conditions where the fibroblast cell would normally proliferate. A control assay may be performed in the absence of the compound to be screened and compared to the amount of fibroblast proliferation in the presence of the compound to determine if the compound stimulates proliferation by determining the uptake of ³[H] thymidine in each case. The amount of fibroblast cell proliferation is measured by liquid scintillation chromatography which measures the incorporation of ³[H] thymidine. Both agonist and antagonist compounds may be identified by this procedure.

In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the present invention is incubated with a labeled polypeptide of the present invention in the presence of the compound. The ability of the compound to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger system following interaction of a compound to be screened and the receptor is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to determine if the compound is a potential agonist or antagonist. Such second messenger systems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognostic markers. The molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule. Moreover, the assays can discover agents which may inhibit or enhance the production of the polypeptides of the invention from suitably manipulated cells or tissues.

Also, one could identify molecules that bind a polypeptide of the invention experimentally by using the beta-pleated sheet regions contained in the polypeptide sequence of the protein. Accordingly, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of each beta pleated sheet regions in a disclosed polypeptide sequence. Additional embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, any combination or all of contained in the polypeptide sequences of the invention. Additional preferred embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, the amino acid sequence of each of the beta pleated sheet regions in one of the polypeptide sequences of the invention. Additional embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, any combination or all of the beta pleated sheet regions in one of the polypeptide sequences of the invention.

Therefore, the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the present invention; and (b) determining if binding has occurred. Moreover, the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the present invention, (b) assaying a biological activity, and (b) determining if a biological activity of the polypeptide has been altered.

Targeted Delivery

In another embodiment, the invention provides a method of delivering compositions to targeted cells expressing a receptor for a polypeptide of the invention, or cells expressing a cell bound form of a polypeptide of the invention.

As discussed herein, polypeptides or antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions. In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (including antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., polypeptides of the invention or antibodies of the invention) in association with toxins or cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the present invention, or the polynucleotides encoding these polypeptides, to screen for molecules which modify the activities of the polypeptides of the present invention. Such a method would include contacting the polypeptide of the present invention with a selected compound(s) suspected of having antagonist or agonist activity, and assaying the activity of these polypeptides following binding.

This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the present invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the present invention. These methods comprise contacting such an agent with a polypeptide of the present invention or a fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or a fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the present invention.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the present invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is incorporated herein by reference herein. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptides of the present invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the present invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic epitopes with a polypeptide of the invention.

Antisense And Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:X, or the complementary strand thereof, and/or to cDNA sequences contained in cDNA ATCC™ Deposit No:Z identified for example, in Table 1A. In one embodiment, antisense sequence is generated internally, by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, J., Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoR1 site on the 5 end and a HindIII site on the 3 end. Next, the pair of oligonucleotides is heated at 90° C. for one minute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5, 10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes the polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.

In one embodiment, the antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding the polypeptide of the present invention or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a gene of the present invention. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the larger the hybridizing nucleic acid, the more base mismatches with a RNA it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335. Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions of polynucleotide sequences described herein could be used in an antisense approach to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5′-, 3′- or coding region of mRNA of the present invention, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2′-O-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.

While antisense nucleotides complementary to the coding region sequence could be used, those complementary to the transcribed untranslated region are most preferred.

Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within the nucleotide sequence of SEQ ID NO:X. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells which express in vivo. DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing.

The antagonist/agonist may also be employed to treat the diseases described herein.

Thus, the invention provides a method of treating disorders or diseases, including but not limited to the disorders or diseases listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention.

Binding Peptides and Other Molecules

The invention also encompasses screening methods for identifying polypeptides and nonpolypeptides that bind polypeptides of the invention, and the binding molecules identified thereby. These binding molecules are useful, for example, as agonists and antagonists of the polypeptides of the invention. Such agonists and antagonists can be used, in accordance with the invention, in the therapeutic embodiments described in detail, below.

This method comprises the steps of:

a. contacting polypeptides of the invention with a plurality of molecules; and

b. identifying a molecule that binds the polypeptides of the invention.

The step of contacting the polypeptides of the invention with the plurality of molecules may be effected in a number of ways. For example, one may contemplate immobilizing the polypeptides on a solid support and bringing a solution of the plurality of molecules in contact with the immobilized polypeptides. Such a procedure would be akin to an affinity chromatographic process, with the affinity matrix being comprised of the immobilized polypeptides of the invention. The molecules having a selective affinity for the polypeptides can then be purified by affinity selection. The nature of the solid support, process for attachment of the polypeptides to the solid support, solvent, and conditions of the affinity isolation or selection are largely conventional and well known to those of ordinary skill in the art.

Alternatively, one may also separate a plurality of polypeptides into substantially separate fractions comprising a subset of or individual polypeptides. For instance, one can separate the plurality of polypeptides by gel electrophoresis, column chromatography, or like method known to those of ordinary skill for the separation of polypeptides. The individual polypeptides can also be produced by a transformed host cell in such a way as to be expressed on or about its outer surface (e.g., a recombinant phage). Individual isolates can then be “probed” by the polypeptides of the invention, optionally in the presence of an inducer should one be required for expression, to determine if any selective affinity interaction takes place between the polypeptides and the individual clone. Prior to contacting the polypeptides with each fraction comprising individual polypeptides, the polypeptides could first be transferred to a solid support for additional convenience. Such a solid support may simply be a piece of filter membrane, such as one made of nitrocellulose or nylon. In this manner, positive clones could be identified from a collection of transformed host cells of an expression library, which harbor a DNA construct encoding a polypeptide having a selective affinity for polypeptides of the invention. Furthermore, the amino acid sequence of the polypeptide having a selective affinity for the polypeptides of the invention can be determined directly by conventional means or the coding sequence of the DNA encoding the polypeptide can frequently be determined more conveniently. The primary sequence can then be deduced from the corresponding DNA sequence. If the amino acid sequence is to be determined from the polypeptide itself, one may use microsequencing techniques. The sequencing technique may include mass spectroscopy.

In certain situations, it may be desirable to wash away any unbound polypeptides from a mixture of the polypeptides of the invention and the plurality of polypeptides prior to attempting to determine or to detect the presence of a selective affinity interaction. Such a wash step may be particularly desirable when the polypeptides of the invention or the plurality of polypeptides are bound to a solid support.

The plurality of molecules provided according to this method may be provided by way of diversity libraries, such as random or combinatorial peptide or nonpeptide libraries which can be screened for molecules that specifically bind polypeptides of the invention. Many libraries are known in the art that can be used, e.g., chemically synthesized libraries, recombinant (e.g., phage display libraries), and in vitro translation-based libraries. Examples of chemically synthesized libraries are described in Fodor et al., 1991, Science 251:767-773; Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature 354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et al., 1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques 13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott and Smith, 1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406; Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra, 1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65; and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

By way of examples of nonpeptide libraries, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).

The variety of non-peptide libraries that are useful in the present invention is great. For example, Ecker and Crooke, 1995, Bio/Technology 13:351-360 list benzodiazepines, hydantoins, piperazinediones, biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids, acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, and oxazolones as among the chemical species that form the basis of various libraries.

Non-peptide libraries can be classified broadly into two types: decorated monomers and oligomers. Decorated monomer libraries employ a relatively simple scaffold structure upon which a variety functional groups is added. Often the scaffold will be a molecule with a known useful pharmacological activity. For example, the scaffold might be the benzodiazepine structure.

Non-peptide oligomer libraries utilize a large number of monomers that are assembled together in ways that create new shapes that depend on the order of the monomers. Among the monomer units that have been used are carbamates, pyrrolinones, and morpholinos. Peptoids, peptide-like oligomers in which the side chain is attached to the alpha amino group rather than the alpha carbon, form the basis of another version of non-peptide oligomer libraries. The first non-peptide oligomer libraries utilized a single type of monomer and thus contained a repeating backbone. Recent libraries have utilized more than one monomer, giving the libraries added flexibility.

Screening the libraries can be accomplished by any of a variety of commonly known methods. See, e.g., the following references, which disclose screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390; Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell 76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992, Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all to Ladner et al.; Rebar and Pabo, 1993, Science 263:671-673; and CT Publication No. WO 94/18318.

In a specific embodiment, screening to identify a molecule that binds polypeptides of the invention can be carried out by contacting the library members with polypeptides of the invention immobilized on a solid phase and harvesting those library members that bind to the polypeptides of the invention. Examples of such screening methods, termed “panning” techniques are described by way of example in Parmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and in references cited herein.

In another embodiment, the two-hybrid system for selecting interacting proteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used to identify molecules that specifically bind to polypeptides of the invention.

Where the binding molecule is a polypeptide, the polypeptide can be conveniently selected from any peptide library, including random peptide libraries, combinatorial peptide libraries, or biased peptide libraries. The term “biased” is used herein to mean that the method of generating the library is manipulated so as to restrict one or more parameters that govern the diversity of the resulting collection of molecules, in this case peptides.

Thus, a truly random peptide library would generate a collection of peptides in which the probability of finding a particular amino acid at a given position of the peptide is the same for all 20 amino acids. A bias can be introduced into the library, however, by specifying, for example, that a lysine occur every fifth amino acid or that positions 4, 8, and 9 of a decapeptide library be fixed to include only arginine. Clearly, many types of biases can be contemplated, and the present invention is not restricted to any particular bias. Furthermore, the present invention contemplates specific types of peptide libraries, such as phage displayed peptide libraries and those that utilize a DNA construct comprising a lambda phage vector with a DNA insert.

As mentioned above, in the case of a binding molecule that is a polypeptide, the polypeptide may have about 6 to less than about 60 amino acid residues, preferably about 6 to about 10 amino acid residues, and most preferably, about 6 to about 22 amino acids. In another embodiment, a binding polypeptide has in the range of 15-100 amino acids, or 20-50 amino acids.

The selected binding polypeptide can be obtained by chemical synthesis or recombinant expression.

Other Activities

A polypeptide, polynucleotide, agonist, or antagonist of the present invention, as a result of the ability to stimulate vascular endothelial cell growth, may be employed in treatment for stimulating re-vascularization of ischemic tissues due to various disease conditions such as thrombosis, arteriosclerosis, and other cardiovascular conditions. The polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed to stimulate angiogenesis and limb regeneration, as discussed above.

A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed for treating wounds due to injuries, burns, post-operative tissue repair, and ulcers since they are mitogenic to various cells of different origins, such as fibroblast cells and skeletal muscle cells, and therefore, facilitate the repair or replacement of damaged or diseased tissue.

A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed stimulate neuronal growth and to treat and prevent neuronal damage which occurs in certain neuronal disorders or neuro-degenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complex. A polypeptide, polynucleotide, agonist, or antagonist of the present invention may have the ability to stimulate chondrocyte growth, therefore, they may be employed to enhance bone and periodontal regeneration and aid in tissue transplants or bone grafts.

A polypeptide, polynucleotide, agonist, or antagonist of the present invention may be also be employed to prevent skin aging due to sunburn by stimulating keratinocyte growth.

A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed for preventing hair loss, since FGF family members activate hair-forming cells and promotes melanocyte growth. Along the same lines, a polypeptide, polynucleotide, agonist, or antagonist of the present invention may be employed to stimulate growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.

A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed to maintain organs before transplantation or for supporting cell culture of primary tissues. A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be employed for inducing tissue of mesodermal origin to differentiate in early embryos.

A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells, besides, as discussed above, hematopoietic lineage.

A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery). Similarly, a polypeptide, polynucleotide, agonist, or antagonist of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.

Polypeptides, polynucleotides, agonists, or antagonists of the present invention may be used to treat weight disorders, including but not limited to, obesity, cachexia, wasting disease, anorexia, and bulimia.

A polypeptide, polynucleotide, agonist, or antagonist of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, circadian rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, reproductive capabilities (preferably by Activin or Inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.

A polypeptide, polynucleotide, agonist, or antagonist of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.

The above-recited applications have uses in a wide variety of hosts. Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.

Other Preferred Embodiments

Other preferred embodiments of the claimed invention include an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 50 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC™ Deposit No:Z. X can be any integer as defined in Table 1A. Also preferred is the above nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the Clone Sequence and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1A. Further preferred is the above nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the Start Codon and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1A. Similarly preferred is the above nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of SEQ ID NO:X in the range of positions beginning with the nucleotide at about the position of the 5′ Nucleotide of the First Amino Acid of the Signal Peptide and ending with the nucleotide at about the position of the 3′ Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in Table 1A.

Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of the portion of SEQ ID NO:X as defined in column 5, “ORF (From-To)”, in Table 1B.1.

Also preferred is a nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of the portion of SEQ ID NO:X as defined in columns 8 and 9, “NT From” and “NT To” respectively, in Table 2.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC™ Deposit No:Z.

Further preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC™ Deposit No:Z.

A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of the portion of SEQ ID NO:X defined in column 5, “ORF (From-To)”, in Table 1B.1.

A further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of the portion of SEQ ID NO:X defined in columns 8 and 9, “NT From” and “NT To”, respectively, in Table 2.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC™ Deposit No:Z.

Also preferred is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto, the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto, and/or cDNA contained in ATCC™ Deposit No:Z, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.

Also preferred is a composition of matter comprising a DNA molecule which comprises the cDNA contained in ATCC™ Deposit No:Z.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides of the cDNA sequence contained in ATCC™ Deposit No:Z.

Also preferred is an isolated nucleic acid molecule, wherein said sequence of at least 50 contiguous nucleotides is included in the nucleotide sequence of an open reading frame sequence encoded by cDNA contained in ATCC™ Deposit No:Z.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 150 contiguous nucleotides in the nucleotide sequence encoded by cDNA contained in ATCC™ Deposit No:Z.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to sequence of at least 500 contiguous nucleotides in the nucleotide sequence encoded by cDNA contained in ATCC™ Deposit No:Z.

A further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence encoded by cDNA contained in ATCC™ Deposit No:Z.

A further preferred embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence encoded by cDNA contained in ATCC™ Deposit No:Z; which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence.

Also preferred is the above method wherein said step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group. Similarly, also preferred is the above method wherein said step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

A further preferred embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence of the cDNA contained in ATCC™ Deposit No:Z.

The method for identifying the species, tissue or cell type of a biological sample can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto; or the cDNA contained in ATCC™ Deposit No:Z which encodes a protein, wherein the method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence of cDNA contained in ATCC™ Deposit No:Z.

The method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X or the complementary strand thereto; the nucleotide sequence as defined in Table 1B.1 (such as column 5 of Table 1B.1) or columns 8 and 9 of Table 2 or the complementary strand thereto; and a nucleotide sequence encoded by cDNA contained in ATCC™ Deposit No:Z. The nucleic acid molecules can comprise DNA molecules or RNA molecules.

Also preferred is a composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a DNA microarray or “chip” of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 100, 150, 200, 250, 300, 500, 1000, 2000, 3000, or 4000 nucleotide sequences, wherein at least one sequence in said DNA microarray or “chip” is at least 95% identical to a sequence of at least 50 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of SEQ ID NO:X wherein X is any integer as defined in Table 1A and/or Tables 1B.1 and 1B.2; and a nucleotide sequence encoded by a human cDNA clone identified by a cDNA “Clone ID” in Table 1A and/or Tables 1B.1 and 1B.2. Further preferred is the above isolated polypeptide, wherein said sequence of contiguous amino acids is included in the amino acid sequence of SEQ ID NO:Y in the range of positions beginning with the residue at about the position of the First Amino Acid of the Secreted Portion and ending with the residue at about the Last Amino Acid of the Open Reading Frame as set forth for SEQ ID NO:Y in Table 1A.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCC™ Deposit No:Z.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCC™ Deposit No:Z.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCC™ Deposit No:Z.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the complete amino acid sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or a polypeptide encoded by cDNA contained in ATCC™ Deposit No:Z.

Further preferred is an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in the complete amino acid sequence of a polypeptide encoded by contained in ATCC™ Deposit No:Z

Also preferred is a polypeptide wherein said sequence of contiguous amino acids is included in the amino acid sequence of a portion of said polypeptide encoded by cDNA contained in ATCC™ Deposit No:Z; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and/or the polypeptide sequence of SEQ ID NO:Y.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in the amino acid sequence of a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 100 contiguous amino acids in the amino acid sequence of a polypeptide encoded by cDNA contained in ATCC™ Deposit No:Z.

Also preferred is an isolated polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence of a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z.

Further preferred is an isolated antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z.

Further preferred is a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z; which method comprises a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to said sequence of at least 10 contiguous amino acids.

Also preferred is the above method wherein said step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: a polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z.

Also preferred is the above method wherein said step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.

Also preferred is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z.

Also preferred is the above method for identifying the species, tissue or cell type of a biological sample, which method comprises a step of detecting polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the above group.

Also preferred is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a nucleic acid sequence identified in Tables 1A, 1B.1, 1B.2 or 2 encoding a polypeptide, which method comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z.

In any of these methods, the step of detecting said polypeptide molecules includes using an antibody.

Also preferred is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z.

Also preferred is an isolated nucleic acid molecule, wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.

Also preferred is a polypeptide molecule, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z.

Further preferred is a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecules into a vector. Also preferred is the recombinant vector produced by this method. Also preferred is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.

Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a human protein comprising an amino acid sequence selected from the group consisting of: polypeptide sequence of SEQ ID NO:Y; an amino acid sequence of SEQ ID NO:Y beginning with the residue at the position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y wherein Y is an integer set forth in Table 1A and said position of the First Amino Acid of the Secreted Portion of SEQ ID NO:Y is defined in Table 1A; and an amino acid sequence of a secreted portion of a protein encoded by a human cDNA clone identified by a cDNA Clone Identifier in Table 1A and contained in the deposit with ATCC™ Deposit No:Z; a polypeptide encoded by SEQ ID NO:X or the complementary strand thereto; the polypeptide encoded by the nucleotide sequence as defined in columns 8 and 9 of Table 2; and a polypeptide encoded by the cDNA contained in ATCC™ Deposit No:Z. The isolated polypeptide produced by this method is also preferred.

Also preferred is a method of treatment of an individual in need of an increased level of a protein activity, which method comprises administering to such an individual a Therapeutic comprising an amount of an isolated polypeptide, polynucleotide, immunogenic fragment or analogue thereof, binding agent, antibody, or antigen binding fragment of the claimed invention effective to increase the level of said protein activity in said individual.

Also preferred is an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: (a) a polynucleotide fragment of SEQ ID NO:X or a polynucleotide fragment of the cDNA sequence included in ATCC™ Deposit No:Z, which is hybridizable to SEQ ID NO:X; (b) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA sequence included in ATCC™ Deposit No:Z, which is hybridizable to SEQ ID NO:X; (c) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y or a polypeptide domain encoded by the cDNA sequence included in ATCC™ Deposit No:Z, which is hybridizable to SEQ ID NO:X; (d) a polynucleotide encoding a polypeptide epitope of SEQ ID NO:Y or a polypeptide epitope encoded by the cDNA sequence included in ATCC™ Deposit No:Z, which is hybridizable to SEQ ID NO:X; (e) a polynucleotide encoding a polypeptide of SEQ ID NO:Y or the cDNA sequence included in ATCC™ Deposit No:Z, which is hybridizable to SEQ ID NO:X, having biological activity; (f) a polynucleotide which is a variant of SEQ ID NO:X; (g) a polynucleotide which is an allelic variant of SEQ ID NO:X; (h) a polynucleotide which encodes a species homologue of the SEQ ID NO:Y; and (i) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h), wherein said polynucleotide does not hybridize under stringent conditions to a nucleic acid molecule having a nucleotide sequence of only A residues or of only T residues.

Also preferred is the isolated nucleic acid molecule as described in preferred embodiment I, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a secreted protein. The nucleotide sequence encoding a secreted protein may further comprise sequential nucleotide deletions from either the C-terminus or the N-terminus.

Also preferred is the isolated nucleic acid molecule as described in preferred embodiment I, wherein the polynucleotide fragment comprises a nucleotide sequence encoding the sequence identified as SEQ ID NO:Y or the polypeptide encoded by the cDNA sequence included in ATCC™ Deposit No:Z, which is hybridizable to SEQ ID NO:X. The nucleotide sequence encoding the sequence identified as SEQ ID NO:Y or the polypeptide encoded by the cDNA sequence included in ATCC™ Deposit No:Z may further comprise sequential nucleotide deletions from either the C-terminus or the N-terminus.

Also preferred is the isolated nucleic acid molecule as described in preferred embodiment I, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO:X or the cDNA sequence included in ATCC™ Deposit No:Z, which is hybridizable to SEQ ID NO:X.

Also preferred is a recombinant vector comprising the isolated nucleic acid molecule described in preferred embodiment I.

Also preferred is a recombinant host cell comprising the isolated nucleic acid molecule described in preferred embodiment I and the method of making the recombinant host cell. The recombinant host cell may further comprise vector sequences.

Also preferred is a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising determining the presence or absence of a mutation in the polynucleotide described in preferred embodiment I and diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or absence of the mutation.

An isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: (a) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence included in ATCC™ Deposit No:Z; (b) a polypeptide fragment of SEQ ID NO:Y or the encoded sequence included in ATCC™ Deposit No:Z, having biological activity; (c) a polypeptide domain of SEQ ID NO:Y or the encoded sequence included in ATCC™ Deposit No:Z; (d) a polypeptide epitope of SEQ ID NO:Y or the encoded sequence included in ATCC™ Deposit No:Z; (e) a secreted form of SEQ ID NO:Y or the encoded sequence included in ATCC™ Deposit No:Z; (f) a full length protein of SEQ ID NO:Y or the encoded sequence included in ATCC™ Deposit No:Z; (g) a variant of SEQ ID NO:Y; (h) an allelic variant of SEQ ID NO:Y; and (i) a species homologue of SEQ ID NO:Y.

Also preferred is the isolated polypeptide as described in preferred embodiment II, wherein the secreted form or the full length protein comprises sequential amino acid deletions from either the C-terminus or the N-terminus.

Also preferred is an isolated antibody that binds specifically to the isolated polypeptide described in preferred embodiment II.

Also preferred is a recombinant host cell that expresses the isolated polypeptide described in preferred embodiment II.

Also preferred is a method of making an isolated polypeptide that comprises culturing the recombinant host cells that expresses the isolated polypeptide described in preferred embodiment II and recovering said polypetide. Further envisioned is the polypeptide produced by this method.

Also preferred is a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject comprising: (a) determining the presence or amount of expression of the polypeptide described in preferred embodiment II in a biological sample; and (b) diagnosing a pathological condition or a susceptibility to a pathological condition based on the presence or amount of expression of the polypeptide.

Also preferred is a method for identifying a binding partner to the polypeptide described in preferred embodiment II comprising: (a) contacting the polypeptide with a binding partner; and (b) determining whether the binding partner effects an activity of the polypeptide

The gene corresponding to the cDNA sequence of SEQ ID NO:X.

A method of identifying an activity in a biological assay, wherein the method comprises: (a) expressing SEQ ID NO:X in a cell; (b) isolating the supernatent; (c) detecting an activity in a biological assay; and (d) identifying the protein in the supernatent having the activity. Preferred embodiment IV may further include the product produced by the method.

[1241] Also preferred is a method for preventing, treating, or ameliorating a medical condition comprising administering to a mammalian subject a therapeutically effective amount of the polynucleotide described in preferred embodiment I or the polypeptide described in preferred embodiment II.

Also preferred is a method of treatment of an individual in need of a decreased level of a protein activity, which method comprised administering to such an individual a Therapeutic comprising an amount of an isolated polypeptide, polynucleotide, immunogenic fragment or analogue thereof, binding agent, antibody, or antigen binding fragment of the claimed invention effective to decrease the level of said protein activity in said individual.

Also preferred is a method of treatment of an individual in need of a specific delivery of toxic compositions to diseased cells (e.g., tumors, leukemias or lymphomas), which method comprises administering to such an individual a Therapeutic comprising an amount of an isolated polypeptide of the invention, including, but not limited to a binding agent, or antibody of the claimed invention that are associated with toxin or cytotoxic prodrugs.

Having generally described the invention, the same will be more readily understood by reference to the examples disclosed herein, which are provided by way of illustration and are not intended as limiting.

In specific embodiments of the invention, for each “Contig ID” listed in the second column of Table 2, preferably excluded are one or more polynucleotides comprising, or alternatively consisting of, a nucleotide sequence referenced in the fifth column of Table 2 and described by the general formula of a-b, whereas a and b are uniquely determined for the corresponding SEQ ID NO:X referred to in column 3 of Table 2. Further specific embodiments are directed to polynucleotide sequences excluding one, two, three, four, or more of the specific polynucleotide sequences referred to in the fifth column of Table 2. In no way is this listing meant to encompass all of the sequences which may be excluded by the general formula, it is just a representative example. All references available through these accessions are hereby incorporated by reference in their entirety.

Table 6

Table 6 summarizes some of the ATCC™ Deposits, Deposit dates, and ATCC™ designation numbers of deposits made with the ATCC™ in connection with the present application. These deposits were made in addition to those described in the Table 1A.

TABLE 6 ATCC ™ Deposits Deposit Date ATCC ™ Designation Number LP01, LP02, LP03, May 20, 1997 209059, 209060, 209061, LP04, LP05, LP06, 209062, 209063, 209064, LP07, LP08, LP09, 209065, 209066, 209067, LP10, LP11 209068, 209069 LP12 Jan. 12, 1998 209579 LP13 Jan. 12, 1998 209578 LP14 Jul. 16, 1998 203067 LP15 Jul. 16, 1998 203068 LP16 Feb. 1, 1999 203609 LP17 Feb. 1, 1999 203610 LP20 Nov. 17, 1998 203485 LP21 Jun. 18, 1999 PTA-252 LP22 Jun. 18, 1999 PTA-253 LP23 Dec. 22, 1999 PTA-1081

EXAMPLES Example 1 Isolation of a Selected cDNA Clone from the Deposited Sample

Each ATCC™ Deposit No:Z is contained in a plasmid vector. Table 1A identifies the vectors used to construct the cDNA library from which each clone was isolated. In many cases, the vector used to construct the library is a phage vector from which a plasmid has been excised. The following correlates the related plasmid for each phage vector used in constructing the cDNA library. For example, where a particular clone is identified in Table 1A as being isolated in the vector “LAMBDA ZAP™,” the corresponding deposited clone is in “pBLUESCRIPT™.”

Vector Used to Construct Library Corresponding Deposited Plasmid LAMBDA ZAP ™ pBLUESCRIPT ™ (pBS) UNI-ZAP ™ XR pBLUESCRIPT ™ (pBS) ZAP EXPRESS ™ pBK lafmid BA plafmid BA pSport1 pSport1 pCMVSport 2.0 pCMVSport 2.0 pCMVSport 3.0 pCMVSport 3.0 pCR ®2.1 pCR ®2.1

Vectors LAMBDA ZAP™ (U.S. Pat. Nos. 5,128,256 and 5,286,636), UNI-ZAP™ XR (U.S. Pat. Nos. 5,128,256 and 5,286,636), ZAP EXPRESS™ (U.S. Pat. Nos. 5,128,256 and 5,286,636), pBLUESCRIPT™ (pBS) (Short, J. M. et al., Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J. M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. et al., Strategies 5:58-61 (1992)) are commercially available from STRATAGENE™ Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla, Calif., 92037. pBS contains an ampicillin resistance gene and pBK contains a neomycin resistance gene. Both can be transformed into E. coli strain XL-1 Blue, also available from STRATAGENE™. pBS comes in 4 forms SK+, SK−, KS+ and KS. The S and K refers to the orientation of the polylinker to the T7 and T3 primer sequences which flank the polylinker region (“S” is for Sad and “K” is for KpnI which are the first sites on each respective end of the linker). “+” or “−” refer to the orientation of the f1 origin of replication (“ori”), such that in one orientation, single stranded rescue initiated from the f1 on generates sense strand DNA and in the other, antisense.

Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtained from LIFE TECHNOLOGIES™, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. All Sport vectors contain an ampicillin resistance gene and may be transformed into E. coli strain DH10B, also available from LIFE TECHNOLOGIES™. (See, for instance, Gruber, C. E., et al., Focus 15:59 (1993)). Vector lafmid BA (Bento Soares, Columbia University, NY) contains an ampicillin resistance gene and can be transformed into E. coli strain XL-1 Blue. Vector pCR®2.1, which is available from Invitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains an ampicillin resistance gene and may be transformed into E. coli strain DH10B, available from LIFE TECHNOLOGIES™. (See, for instance, Clark, J. M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al., Bio/Technology 9: (1991)). Preferably, a polynucleotide of the present invention does not comprise the phage vector sequences identified for the particular clone in Table 1A, as well as the corresponding plasmid vector sequences designated above.

The deposited material in the sample assigned the ATCC™ Deposit Number cited by reference to Tables 1A, 6, and 7 for any given cDNA clone also may contain one or more additional plasmids, each comprising a cDNA clone different from that given clone. Thus, deposits sharing the same ATCC™ Deposit Number contain at least a plasmid for each ATCC™ Deposit No:Z.

TABLE 7 ATCC ™ Libraries owned by Catalog Catalog Description Vector Deposit HUKA HUKB HUKC HUKD Human Uterine Cancer Lambda ZAP II LP01 HUKE HUKF HUKG HCNA HCNB Human Colon Lambda Zap II LP01 HFFA Human Fetal Brain, random Lambda Zap II LP01 primed HTWA Resting T-Cell Lambda ZAP II LP01 HBQA Early Stage Human Brain, Lambda ZAP II LP01 random primed HLMB HLMF HLMG HLMH breast lymph node CDNA Lambda ZAP II LP01 HLMI HLMJ HLMM HLMN library HCQA HCQB human colon cancer Lamda ZAP II LP01 HMEA HMEC HMED Human Microvascular Lambda ZAP II LP01 HMEE HMEF HMEG HMEI Endothelial Cells, fract. A HMEJ HMEK HMEL HUSA HUSC Human Umbilical Vein Lambda ZAP II LP01 Endothelial Cells, fract. A HLQA HLQB Hepatocellular Tumor Lambda ZAP II LP01 HHGA HHGB HHGC HHGD Hemangiopericytoma Lambda ZAP II LP01 HSDM Human Striatum Depression, Lambda ZAP II LP01 re-rescue HUSH H Umbilical Vein Endothelial Lambda ZAP II LP01 Cells, frac A, re-excision HSGS Salivary gland, subtracted Lambda ZAP II LP01 HFXA HFXB HFXC HFXD Brain frontal cortex Lambda ZAP II LP01 HFXE HFXF HFXG HFXH HPQA HPQB HPQC PERM TF274 Lambda ZAP II LP01 HFXJ HFXK Brain Frontal Cortex, re- Lambda ZAP II LP01 excision HCWA HCWB HCWC CD34 positive cells (Cord ZAP Express LP02 HCWD HCWE HCWF Blood) HCWG HCWH HCWI HCWJ HCWK HCUA HCUB HCUC CD34 depleted Buffy Coat ZAP Express LP02 (Cord Blood) HRSM A-14 cell line ZAP Express LP02 HRSA Al-CELL LINE ZAP Express LP02 HCUD HCUE HCUF HCUG CD34 depleted Buffy Coat ZAP Express LP02 HCUH HCUI (Cord Blood), re-excision HBXE HBXF HBXG H. Whole Brain #2, re- ZAP Express LP02 excision HRLM L8 cell line ZAP Express LP02 HBXA HBXB HBXC HBXD Human Whole Brain #2 - ZAP Express LP02 Oligo dT > 1.5Kb HUDA HUDB HUDC Testes ZAP Express LP02 HHTM HHTN HHTO H. hypothalamus, frac A; re- ZAP Express LP02 excision HHTL H. hypothalamus, frac A ZAP Express LP02 HASA HASD Human Adult Spleen Uni-ZAP XR LP03 HFKC HFKD HFKE HFKF Human Fetal Kidney Uni-ZAP XR LP03 HFKG HE8A HE8B HE8C HE8D Human 8 Week Whole Uni-ZAP XR LP03 HE8E HE8F HE8M HE8N Embryo HGBA HGBD HGBE HGBF Human Gall Bladder Uni-ZAP XR LP03 HGBG HGBH HGBI HLHA HLHB HLHC HLHD Human Fetal Lung III Uni-ZAP XR LP03 HLHE HLHF HLHG HLHH HLHQ HPMA HPMB HPMC HPMD Human Placenta Uni-ZAP XR LP03 HPME HPMF HPMG HPMH HPRA HPRB HPRC HPRD Human Prostate Uni-ZAP XR LP03 HSIA HSIC HSID HSIE Human Adult Small Intestine Uni-ZAP XR LP03 HTEA HTEB HTEC HTED Human Testes Uni-ZAP XR LP03 HTEE HTEF HTEG HTEH HTEI HTEJ HTEK HTPA HTPB HTPC HTPD Human Pancreas Tumor Uni-ZAP XR LP03 HTPE HTTA HTTB HTTC HTTD Human Testes Tumor Uni-ZAP XR LP03 HTTE HTTF HAPA HAPB HAPC HAPM Human Adult Pulmonary Uni-ZAP XR LP03 HETA HETB HETC HETD Human Endometrial Tumor Uni-ZAP XR LP03 HETE HETF HETG HETH HETI HHFB HHFC HHFD HHFE Human Fetal Heart Uni-ZAP XR LP03 HHFF HHFG HHFH HHFI HHPB HHPC HHPD HHPE Human Hippocampus Uni-ZAP XR LP03 HHPF HHPG HHPH HCE1 HCE2 HCE3 HCE4 Human Cerebellum Uni-ZAP XR LP03 HCE5 HCEB HCEC HCED HCEE HCEF HCEG HUVB HUVC HUVD HUVE Human Umbilical Vein, Uni-ZAP XR LP03 Endo. remake HSTA HSTB HSTC HSTD Human Skin Tumor Uni-ZAP XR LP03 HTAA HTAB HTAC HTAD Human Activated T-Cells Uni-ZAP XR LP03 HTAE HFEA HFEB HFEC Human Fetal Epithelium Uni-ZAP XR LP03 (Skin) HJPA HJPB HJPC HJPD HUMAN JURKAT Uni-ZAP XR LP03 MEMBRANE BOUND POLYSOMES HESA Human epithelioid sarcoma Uni-Zap XR LP03 HLTA HLTB HLTC HLTD Human T-Cell Lymphoma Uni-ZAP XR LP03 HLTE HLTF HFTA HFTB HFTC HFTD Human Fetal Dura Mater Uni-ZAP XR LP03 HRDA HRDB HRDC HRDD Human Rhabdomyosarcoma Uni-ZAP XR LP03 HRDE HRDF HCAA HCAB HCAC Cem cells cyclohexamide Uni-ZAP XR LP03 treated HRGA HRGB HRGC HRGD Raji Cells, cyclohexamide Uni-ZAP XR LP03 treated HSUA HSUB HSUC HSUM Supt Cells, cyclohexamide Uni-ZAP XR LP03 treated HT4A HT4C HT4D Activated T-Cells, 12 hrs. Uni-ZAP XR LP03 HE9A HE9B HE9C HE9D Nine Week Old Early Stage Uni-ZAP XR LP03 HE9E HE9F HE9G HE9H Human HE9M HE9N HATA HATB HATC HATD Human Adrenal Gland Tumor Uni-ZAP XR LP03 HATE HT5A Activated T-Cells, 24 hrs. Uni-ZAP XR LP03 HFGA HFGM Human Fetal Brain Uni-ZAP XR LP03 HNEA HNEB HNEC HNED Human Neutrophil Uni-ZAP XR LP03 HNEE HBGB HBGD Human Primary Breast Uni-ZAP XR LP03 Cancer HBNA HBNB Human Normal Breast Uni-ZAP XR LP03 HCAS Cem Cells, cyclohexamide Uni-ZAP XR LP03 treated, subtra HHPS Human Hippocampus, pBS LP03 subtracted HKCS HKCU Human Colon Cancer, pBS LP03 subtracted HRGS Raji cells, cyclohexamide pBS LP03 treated, subtracted HSUT Supt cells, cyclohexamide pBS LP03 treated, differentially expressed HT4S Activated T-Cells, 12 hrs, Uni-ZAP XR LP03 subtracted HCDA HCDB HCDC HCDD Human Chondrosarcoma Uni-ZAP XR LP03 HCDE HOAA HOAB HOAC Human Osteosarcoma Uni-ZAP XR LP03 HTLA HTLB HTLC HTLD Human adult testis, large Uni-ZAP XR LP03 HTLE HTLF inserts HLMA HLMC HLMD Breast Lymph node cDNA Uni-ZAP XR LP03 library H6EA H6EB H6EC HL-60, PMA 4H Uni-ZAP XR LP03 HTXA HTXB HTXC HTXD Activated T-Cell Uni-ZAP XR LP03 HTXE HTXF HTXG HTXH (l2 hs)/Thiouridine labelledEco HNFA HNFB HNFC HNFD Human Neutrophil, Activated Uni-ZAP XR LP03 HNFE HNFF HNFG HNFH HNFJ HTOB HTOC HUMAN TONSILS, Uni-ZAP XR LP03 FRACTION 2 HMGB Human OB MG63 control Uni-ZAP XR LP03 fraction I HOPB Human OB HOS control Uni-ZAP XR LP03 fraction I HORB Human OB HOS treated (10 Uni-ZAP XR LP03 nM E2) fraction I HSVA HSVB HSVC Human Chronic Synovitis Uni-ZAP XR LP03 HROA HUMAN STOMACH Uni-ZAP XR LP03 HBJA HBJB HBJC HBJD HUMAN B CELL Uni-ZAP XR LP03 HBJE HBJF HBJG HBJH LYMPHOMA HBJI HBJJ HBJK HCRA HCRB HCRC human corpus colosum Uni-ZAP XR LP03 HODA HODB HODC HODD human ovarian cancer Uni-ZAP XR LP03 HDSA Dermatofibrosarcoma Uni-ZAP XR LP03 Protuberance HMWA HMWB HMWC Bone Marrow Cell Line Uni-ZAP XR LP03 HMWD HMWE HMWF (RS4;11) HMWG HMWH HMWI HMWJ HSOA stomach cancer (human) Uni-ZAP XR LP03 HERA SKIN Uni-ZAP XR LP03 HMDA Brain-medulloblastoma Uni-ZAP XR LP03 HGLA HGLB HGLD Glioblastoma Uni-ZAP XR LP03 HEAA H. Atrophic Endometrium Uni-ZAP XR LP03 HBCA HBCB H. Lymph node breast Cancer Uni-ZAP XR LP03 HPWT Human Prostate BPH, re- Uni-ZAP XR LP03 excision HFVG HFVH HFVI Fetal Liver, subtraction II pBS LP03 HNFI Human Neutrophils, pBS LP03 Activated, re-excision HBMB HBMC HBMD Human Bone Marrow, re- pBS LP03 excision HKML HKMM HKMN H. Kidney Medulla, re- pBS LP03 excision HKIX HKIY H. Kidney Cortex, subtracted pBS LP03 HADT H. Amygdala Depression, pBS LP03 subtracted H6AS HI-60, untreated, subtracted Uni-ZAP XR LP03 H6ES HL-60, PMA 4H, subtracted Uni-ZAP XR LP03 H6BS HL-60, RA 4h, Subtracted Uni-ZAP XR LP03 H6CS HL-60, PMA 1d, subtracted Uni-ZAP XR LP03 HTXJ HTXK Activated T- Uni-ZAP XR LP03 cell(12 h)/Thiouridine-re- excision HMSA HMSB HMSC HMSD Monocyte activated Uni-ZAP XR LP03 HMSE HMSF HMSG HMSH HMSI HMSJ HMSK HAGA HAGB HAGC HAGD Human Amygdala Uni-ZAP XR LP03 HAGE HAGF HSRA HSRB HSRE STROMAL - Uni-ZAP XR LP03 OSTEOCLASTOMA HSRD HSRF HSRG HSRH Human Osteoclastoma Uni-ZAP XR LP03 Stromal Cells - unamplified HSQA HSQB HSQC HSQD Stromal cell TF274 Uni-ZAP XR LP03 HSQE HSQF HSQG HSKA HSKB HSKC HSKD Smooth muscle, serum treated Uni-ZAP XR LP03 HSKE HSKF HSKZ HSLA HSLB HSLC HSLD Smooth muscle,control Uni-ZAP XR LP03 HSLE HSLF HSLG HSDA HSDD HSDE HSDF Spinal cord Uni-ZAP XR LP03 HSDG HSDH HPWS Prostate-BPH subtracted II pBS LP03 HSKW HSKX HSKY Smooth Muscle- HASTE pBS LP03 normalized HFPB HFPC HFPD H. Frontal cortex, epileptic; Uni-ZAP XR LP03 reexcision HSDI HSDJ HSDK Spinal Cord, re-excision Uni-ZAP XR LP03 HSKN HSKO Smooth Muscle Serum pBS LP03 Treated, Norm HSKG HSKH HSKI Smooth muscle, serum pBS LP03 induced, re-exc HFCA HFCB HFCC HFCD Human Fetal Brain Uni-ZAP XR LP04 HFCE HFCF HPTA HPTB HPTD Human Pituitary Uni-ZAP XR LP04 HTHB HTHC HTHD Human Thymus Uni-ZAP XR LP04 HE6B HE6C HEED HE6E Human Whole Six Week Old Uni-ZAP XR LP04 HE6F HE6G HE6S Embryo HSSA HSSB HSSC HSSD Human Synovial Sarcoma Uni-ZAP XR LP04 HSSE HSSF HSSG HSSH HSSI HSSJ HSSK HE7T 7 Week Old Early Stage Uni-ZAP XR LP04 Human, subtracted HEPA HEPB HEPC Human Epididymus Uni-ZAP XR LP04 HSNA HSNB HSNC HSNM Human Synovium Uni-ZAP XR LP04 HSNN HPFB HPFC HPFD HPFE Human Prostate Cancer, Uni-ZAP XR LP04 Stage C fraction HE2A HE2D HE2E HE2H 12 Week Old Early Stage Uni-ZAP XR LP04 HE2I HE2M HE2N HE2O Human HE2B HE2C HE2F HE2G 12 Week Old Early Stage Uni-ZAP XR LP04 HE2P HE2Q Human, II HPTS HPTT HPTU Human Pituitary, subtracted Uni-ZAP XR LP04 HAUA HAUB HAUC Amniotic Cells - TNF Uni-ZAP XR LP04 induced HAQA HAQB HAQC HAQD Amniotic Cells - Primary Uni-ZAP XR LP04 Culture HWTA HWTB HWTC wilm's tumor Uni-ZAP XR LP04 HBSD Bone Cancer, re-excision Uni-ZAP XR LP04 HSGB Salivary gland, re-excision Uni-ZAP XR LP04 HSJA HSJB HSJC Smooth muscle-ILb induced Uni-ZAP XR LP04 HSXA HSXB HSXC HSXD Human Substantia Nigra Uni-ZAP XR LP04 HSHA HSHB HSHC Smooth muscle, IL lb induced Uni-ZAP XR LP04 HOUA HOUB HOUC HOUD Adipocytes Uni-ZAP XR LP04 HOUE HPWA HPWB HPWC Prostate BPH Uni-ZAP XR LP04 HPWD HPWE HELA HELB HELC HELD Endothelial cells-control Uni-ZAP XR LP04 HELE HELF HELG HELH HEMA HEMB HEMC Endothelial-induced Uni-ZAP XR LP04 HEMD HEME HEMF HEMG HEMH HBIA HBIB HBIC Human Brain, Striatum Uni-ZAP XR LP04 HHSA HHSB HHSC HHSD Human Uni-ZAP XR LP04 HHSE Hypothalmus, Schizophrenia HNGA HNGB HNGC HNGD neutrophils control Uni-ZAP XR LP04 HNGE HNGF HNGG HNGH HNGI HNGJ HNHA HNHB HNHC HNHD Neutrophils IL-1 and LPS Uni-ZAP XR LP04 HNHE HNHF HNHG HNHH induced HNHI HNHJ HSDB HSDC STRIATUM DEPRESSION Uni-ZAP XR LP04 HHPT Hypothalamus Uni-ZAP XR LP04 HSAT HSAU HSAV HSAW Anergic T-cell Uni-ZAP XR LP04 HSAX HSAY HSAZ HBMS HBMT HBMU Bone marrow Uni-ZAP XR LP04 HBMV HBMW HBMX HOEA HOEB HOEC HOED Osteoblasts Uni-ZAP XR LP04 HOEE HOEF HOEJ HAIA HAIB HAIC HAID Epithelial-TNFa and INF Uni-ZAP XR LP04 HAIE HAIF induced HTGA HTGB HTGC HTGD Apoptotic T-cell Uni-ZAP XR LP04 HMCA HMCB HMCC Macrophage-oxLDL Uni-ZAP XR LP04 HMCD HMCE HMAA HMAB HMAC Macrophage (GM-CSF Uni-ZAP XR LP04 HMAD HMAE HMAF treated) HMAG HPHA Normal Prostate Uni-ZAP XR LP04 HPIA HPIB HPIC LNCAP prostate cell line Uni-ZAP XR LP04 HPJA HPJB HPJC PC3 Prostate cell line Uni-ZAP XR LP04 HOSE HOSF HOSG Human Osteoclastoma, re- Uni-ZAP XR LP04 excision HTGE HTGF Apoptotic T-cell, re-excision Uni-ZAP XR LP04 HMAJ HMAK H Macrophage (GM-CSF Uni-ZAP XR LP04 treated), re-excision HACB HACC HACD Human Adipose Tissue, re- Uni-ZAP XR LP04 excision HFPA H. Frontal Cortex, Epileptic Uni-ZAP XR LP04 HFAA HFAB HFAC HFAD Alzheimer's, spongy change Uni-ZAP XR LP04 HFAE HFAM Frontal Lobe, Dementia Uni-ZAP XR LP04 HMIA HMIB HMIC Human Manic Depression Uni-ZAP XR LP04 Tissue HTSA HTSE HTSF HTSG Human Thymus pBS LP05 HTSH HPBA HPBB HPBC HPBD Human Pineal Gland pBS LP05 HPBE HSAA HSAB HSAC HSA 172 Cells pBS LP05 HSBA HSBB HSBC HSBM HSC172 cells pBS LP05 HJAA HJAB HJAC HJAD Jurkat T-cell G1 phase pBS LP05 HJBA HJBB HJBC HJBD Jurkat T-Cell, S phase pBS LP05 HAFA HAFB Aorta endothelial cells + pBS LP05 TNF-a HAWA HAWB HAWC Human White Adipose pBS LP05 HTNA HTNB Human Thyroid pBS LP05 HONA Normal Ovary, pBS LP05 Premenopausal HARA HARB Human Adult Retina pBS LP05 HLJA HUB Human Lung pCMVSport 1 LP06 HOFM HOFN HOFO H. Ovarian Tumor, II, pCMVSport 2.0 LP07 OV5232 HOGA HOGB HOGC OV Oct. 3, 1995 pCMVSport 2.0 LP07 HCGL CD34+ cells, II pCMVSport 2.0 LP07 HDLA Hodgkin's Lymphoma I pCMVSport 2.0 LP07 HDTA HDTB HDTC HDTD Hodgkin's Lymphoma II pCMVSport 2.0 LP07 HDTE HKAA HKAB HKAC HKAD Keratinocyte pCMVSport2.0 LP07 HKAE HKAF HKAG HKAH HCIM CAPFINDER, Crohn's pCMVSport 2.0 LP07 Disease, lib 2 HKAL Keratinocyte, lib 2 pCMVSport2.0 LP07 HKAT Keratinocyte, lib 3 pCMVSport2.0 LP07 HNDA Nasal polyps pCMVSport2.0 LP07 HDRA H. Primary Dendritic Cells, pCMVSport2.0 LP07 lib 3 HOHA HOHB HOHC Human Osteoblasts II pCMVSport2.0 LP07 HLDA HLDB HLDC Liver, Hepatoma pCMVSport3.0 LP08 HLDN HLDO HLDP Human Liver, normal pCMVSport3.0 LP08 HMTA pBMC stimulated w/ poly I/C pCMVSport3.0 LP08 HNTA NTERA2, control pCMVSport3.0 LP08 HDPA HDPB HDPC HDPD Primary Dendritic Cells, lib 1 pCMVSport3.0 LP08 HDPF HDPG HDPH HDPI HDPJ HDPK HDPM HDPN HDPO HDPP Primary Dendritic cells, frac 2 pCMVSport3.0 LP08 HMUA HMUB HMUC Myoloid Progenitor Cell Line pCMVSport3.0 LP08 HHEA HHEB HHEC HHED T Cell helper I pCMVSport3.0 LP08 HHEM HHEN HHEO HHEP T cell helper II pCMVSport3.0 LP08 HEQA HEQB HEQC Human endometrial stromal pCMVSport3.0 LP08 cells HJMA HJMB Human endometrial stromal pCMVSport3.0 LP08 cells-treated with progesterone HSWA HSWB HSWC Human endometrial stromal pCMVSport3.0 LP08 cells-treated with estradiol HSYA HSYB HSYC Human Thymus Stromal Cells pCMVSport3.0 LP08 HLWA HLWB HLWC Human Placenta pCMVSport3.0 LP08 HRAA HRAB HRAC Rejected Kidney, lib 4 pCMVSport3.0 LP08 HMTM PCR, pBMC I/C treated PCRII LP09 HMJA H. Meniingima, M6 pSport 1 LP10 HMKA HMKB HMKC H. Meningima, M1 pSport 1 LP10 HMKD HMKE HUSG HUSI Human umbilical vein pSport 1 LP10 endothelial cells, IL-4 induced HUSX HUSY Human Umbilical Vein pSport 1 LP10 Endothelial Cells, uninduced HOFA Ovarian Tumor I, OV5232 pSport 1 LP10 HCFA HCFB HCFC HCFD T-Cell PHA 16 hrs pSport 1 LP10 HCFL HCFM HCFN HCFO T-Cell PHA 24 hrs pSport 1 LP10 HADA HADC HADD HADE Human Adipose pSport 1 LP10 HADF HADG HOVA HOVB HOVC Human Ovary pSport 1 LP10 HTWB HTWC HTWD Resting T-Cell Library, II pSport 1 LP10 HTWE HTWF HMMA Spleen metastic melanoma pSport 1 LP10 HLYA HLYB HLYC HLYD Spleen, Chronic lymphocytic pSport 1 LP10 HLYE leukemia HCGA CD34+ cell, I pSport 1 LP10 HEOM HEON Human Eosinophils pSport 1 LP10 HTDA Human Tonsil, Lib 3 pSport 1 LP10 HSPA Salivary Gland, Lib 2 pSport 1 LP10 HCHA HCHB HCHC Breast Cancer cell line, MDA pSport 1 LP10 36 HCHM HCHN Breast Cancer Cell line, pSport 1 LP10 angiogenic HCIA Crohn's Disease pSport 1 LP10 HDAA HDAB HDAC HEL cell line pSport 1 LP10 HABA Human Astrocyte pSport 1 LP10 HUFA HUFB HUFC Ulcerative Colitis pSport 1 LP10 HNTM NTERA2 + retinoic acid, 14 pSport 1 LP10 days HDQA Primary Dendritic pSport 1 LP10 cells, CapFinder2, frac 1 HDQM Primary Dendritic Cells, pSport 1 LP10 CapFinder, frac 2 HLDX Human Liver, pSport 1 LP10 normal, CapFinder HULA HULB HULC Human Dermal Endothelial pSport1 LP10 Cells, untreated HUMA Human Dermal Endothelial pSport1 LP10 cells, treated HCJA Human Stromal Endometrial pSport1 LP10 fibroblasts, untreated HCJM Human Stromal endometrial pSport1 LP10 fibroblasts, treated w/ estradiol HEDA Human Stromal endometrial pSport1 LP10 fibroblasts, treated with progesterone HFNA Human ovary tumor cell pSport1 LP10 OV350721 HKGA HKGB HKGC HKGD Merkel Cells pSport1 LP10 HISA HISB HISC Pancreas Islet Cell Tumor pSport1 LP10 HLSA Skin, burned pSport1 LP10 HBZA Prostate, BPH, Lib 2 pSport 1 LP10 HBZS Prostate BPH, Lib 2, pSport 1 LP10 subtracted HFIA HFIB HFIC Synovial Fibroblasts (control) pSport 1 LP10 HFIH HFII HFIJ Synovial hypoxia pSport 1 LP10 HFIT HFIU HFIV Synovial IL-1/TNF stimulated pSport 1 LP10 HGCA Messangial cell, frac 1 pSport1 LP10 HMVA HMVB HMVC Bone Marrow Stromal Cell, pSport1 LP10 untreated HFIX HFIY HFIZ Synovial Fibroblasts pSport1 LP10 (Il1/TNF), subt HFOX HFOY HFOZ Synovial hypoxia-RSF pSport1 LP10 subtracted HMQA HMQB HMQC Human Activated Monocytes Uni-ZAP XR LP11 HMQD HLIA HLIB HLIC Human Liver pCMVSport 1 LP012 HHBA HHBB HHBC HHBD Human Heart pCMVSport 1 LP012 HHBE HBBA HBBB Human Brain pCMVSport 1 LP012 HLJA HLJB HLJC HLJD Human Lung pCMVSport 1 LP012 HLJE HOGA HOGB HOGC Ovarian Tumor pCMVSport 2.0 LP012 HTJM Human Tonsils, Lib 2 pCMVSport 2.0 LP012 HAMF HAMG KMH2 pCMVSport 3.0 LP012 HAJA HAJB HAJC L428 pCMVSport 3.0 LP012 HWBA HWBB HWBC Dendritic cells, pooled pCMVSport 3.0 LP012 HWBD HWBE HWAA HWAB HWAC Human Bone Marrow, pCMVSport 3.0 LP012 HWAD HWAE treated HYAA HYAB HYAC B Cell lymphoma pCMVSport 3.0 LP012 HWHG HWHH HWHI Healing groin wound, 6.5 pCMVSport 3.0 LP012 hours post incision HWHP HWHQ HWHR Healing groin wound; 7.5 pCMVSport 3.0 LP012 hours post incision HARM Healing groin wound - zero hr pCMVSport 3.0 LP012 post-incision (control) HBIM Olfactory epithelium; pCMVSport 3.0 LP012 nasalcavity HWDA Healing Abdomen wound; pCMVSport 3.0 LP012 70&90 min post incision HWEA Healing Abdomen Wound; 15 pCMVSport 3.0 LP012 days post incision HWJA Healing Abdomen pCMVSport 3.0 LP012 Wound; 21&29 days HNAL Human Tongue, frac 2 pSport1 LP012 HMJA H. Meniingima, M6 pSport1 LP012 HMKA HMKB HMKC H. Meningima, M1 pSport1 LP012 HMKD HMKE HOFA Ovarian Tumor I, OV5232 pSport1 LP012 HCFA HCFB HCFC HCFD T-Cell PHA 16 hrs pSport1 LP012 HCFL HCFM HCFN HCFO T-Cell PHA 24 hrs pSport1 LP012 HMMA HMMB HMMC Spleen metastic melanoma pSport1 LP012 HTDA Human Tonsil, Lib 3 pSport1 LP012 HDBA Human Fetal Thymus pSport1 LP012 HDUA Pericardium pSport1 LP012 HBZA Prostate, BPH, Lib 2 pSport1 LP012 HWCA Larynx tumor pSport1 LP012 HWKA Normal lung pSport1 LP012 HSMB Bone marrow stroma, treated pSport1 LP012 HBHM Normal trachea pSport1 LP012 HLFC Human Larynx pSport1 LP012 HLRB Siebben Polyposis pSport1 LP012 HNIA Mammary Gland pSport1 LP012 HNJB Palate carcinoma pSport1 LP012 HNKA Palate normal pSport1 LP012 HMZA Pharynx carcinoma pSport1 LP012 HABG Cheek Carcinoma pSport1 LP012 HMZM Pharynx Carcinoma pSport1 LP012 HDRM Larynx Carcinoma pSport1 LP012 HVAA Pancreas normal PCA4 No pSport1 LP012 HICA Tongue carcinoma pSport1 LP012 HUKA HUKB HUKC HUKD Human Uterine Cancer Lambda ZAP II LP013 HUKE HFFA Human Fetal Brain, random Lambda ZAP II LP013 primed HTUA Activated T-cell labeled with Lambda ZAP II LP013 4-thioluri HBQA Early Stage Human Brain, Lambda ZAP II LP013 random primed HMEB Human microvascular Lambda ZAP II LP013 Endothelial cells, fract. B HUSH Human Umbilical Vein Lambda ZAP II LP013 Endothelial cells, fract. A, re- excision HLQC HLQD Hepatocellular tumor, re- Lambda ZAP II LP013 excision HTWJ HTWK HTWL Resting T-cell, re-excision Lambda ZAP II LP013 HF6S Human Whole 6 week Old pBLUESCRIPT ™ LP013 Embryo (II), subt HHPS Human Hippocampus, pBLUESCRIPT ™ LP013 subtracted HL1S LNCAP, differential pBLUESCRIPT ™ LP013 expression HLHS HLHT Early Stage Human Lung, pBLUESCRIPT ™ LP013 Subtracted HSUS Supt cells, cyclohexamide pBLUESCRIPT ™ LP013 treated, subtracted HSUT Supt cells, cyclohexamide pBLUESCRIPT ™ LP013 treated, differentially expressed HSDS H. Striatum Depression, pBLUESCRIPT ™ LP013 subtracted HPTZ Human Pituitary, Subtracted pBLUESCRIPT ™ LP013 VII HSDX H. Striatum Depression, subt pBLUESCRIPT ™ LP013 II HSDZ H. Striatum Depression, subt pBLUESCRIPT ™ LP013 HPBA HPBB HPBC HPBD Human Pineal Gland pBLUESCRIPT ™ LP013 HPBE SK- HRTA Colorectal Tumor pBLUESCRIPT ™ LP013 SK- HSBA HSBB HSBC HSBM HSC172 cells pBLUESCRIPT ™ LP013 SK- HJAA HJAB HJAC HJAD Jurkat T-cell G1 phase pBLUESCRIPT ™ LP013 SK- HJBA HJBB HJBC HJBD Jurkat T-cell, Si phase pBLUESCRIPT ™ LP013 SK- HTNA HTNB Human Thyroid pBLUESCRIPT ™ LP013 SK- HAHA HAHB Human Adult Heart Uni-ZAP XR LP013 HE6A Whole 6 week Old Embryo Uni-ZAP XR LP013 HFCA HFCB HFCC HFCD Human Fetal Brain Uni-ZAP XR LP013 HFCE HFKC HFKD HFKE HFKF Human Fetal Kidney Uni-ZAP XR LP013 HFKG HGBA HGBD HGBE HGBF Human Gall Bladder Uni-ZAP XR LP013 HGBG HPRA HPRB HPRC HPRD Human Prostate Uni-ZAP XR LP013 HTEA HTEB HTEC HTED Human Testes Uni-ZAP XR LP013 HTEE HTTA HTTB HTTC HTTD Human Testes Tumor Uni-ZAP XR LP013 HTTE HYBA HYBB Human Fetal Bone Uni-ZAP XR LP013 HFLA Human Fetal Liver Uni-ZAP XR LP013 HHFB HHFC HHFD HHFE Human Fetal Heart Uni-ZAP XR LP013 HHFF HUVB HUVC HUVD HUVE Human Umbilical Vein, End. Uni-ZAP XR LP013 remake HTHB HTHC HTHD Human Thymus Uni-ZAP XR LP013 HSTA HSTB HSTC HSTD Human Skin Tumor Uni-ZAP XR LP013 HTAA HTAB HTAC HTAD Human Activated T-cells Uni-ZAP XR LP013 HTAE HFEA HFEB HFEC Human Fetal Epithelium Uni-ZAP XR LP013 (skin) HJPA HJPB HJPC HJPD Human Jurkat Membrane Uni-ZAP XR LP013 Bound Polysomes HESA Human Epithelioid Sarcoma Uni-ZAP XR LP013 HALS Human Adult Liver, Uni-ZAP XR LP013 Subtracted HFTA HFTB HFTC HFTD Human Fetal Dura Mater Uni-ZAP XR LP013 HCAA HCAB HCAC Cem cells, cyclohexamide Uni-ZAP XR LP013 treated HRGA HRGB HRGC HRGD Raji Cells, cyclohexamide Uni-ZAP XR LP013 treated HE9A HE9B HE9C HE9D Nine Week Old Early Stage Uni-ZAP XR LP013 HE9E Human HSFA Human Fibrosarcoma Uni-ZAP XR LP013 HATA HATB HATC HATD Human Adrenal Gland Tumor Uni-ZAP XR LP013 HATE HTRA Human Trachea Tumor Uni-ZAP XR LP013 HE2A HE2D HE2E HE2H 12 Week Old Early Stage Uni-ZAP XR LP013 HE2I Human HE2B HE2C HE2F HE2G 12 Week Old Early Stage Uni-ZAP XR LP013 HE2P Human, II HNEA HNEB HNEC HNED Human Neutrophil Uni-ZAP XR LP013 HNEE HBGA Human Primary Breast Uni-ZAP XR LP013 Cancer HPTS HPTT HPTU Human Pituitary, subtracted Uni-ZAP XR LP013 HMQA HMQB HMQC Human Activated Monocytes Uni-ZAP XR LP013 HMQD HOAA HOAB HOAC Human Osteosarcoma Uni-ZAP XR LP013 HTOA HTOD HTOE HTOF human tonsils Uni-ZAP XR LP013 HTOG HMGB Human OB MG63 control Uni-ZAP XR LP013 fraction I HOPB Human OB HOS control Uni-ZAP XR LP013 fraction I HOQB Human OB HOS treated (1 Uni-ZAP XR LP013 nM E2) fraction I HAUA HAUB HAUC Amniotic Cells - TNF Uni-ZAP XR LP013 induced HAQA HAQB HAQC HAQD Amniotic Cells - Primary Uni-ZAP XR LP013 Culture HROA HROC HUMAN STOMACH Uni-ZAP XR LP013 HBJA HBJB HBJC HBJD HUMAN B CELL Uni-ZAP XR LP013 HBJE LYMPHOMA HODA HODB HODC HODD human ovarian cancer Uni-ZAP XR LP013 HCPA Corpus Callosum Uni-ZAP XR LP013 HSOA stomach cancer (human) Uni-ZAP XR LP013 HERA SKIN Uni-ZAP XR LP013 HMDA Brain-medulloblastoma Uni-ZAP XR LP013 HGLA HGLB HGLD Glioblastoma Uni-ZAP XR LP013 HWTA HWTB HWTC wilm's tumor Uni-ZAP XR LP013 HEAA H. Atrophic Endometrium Uni-ZAP XR LP013 HAPN HAPO HAPP HAPQ Human Adult Pulmonary; re- Uni-ZAP XR LP013 HAPR excision HLTG HLTH Human T-cell lymphoma; re- Uni-ZAP XR LP013 excision HAHC HAHD HAHE Human Adult Heart; re- Uni-ZAP XR LP013 excision HAGA HAGB HAGC HAGD Human Amygdala Uni-ZAP XR LP013 HAGE HSJA HSJB HSJC Smooth muscle-ILb induced Uni-ZAP XR LP013 HSHA HSHB HSHC Smooth muscle, IL1b induced Uni-ZAP XR LP013 HPWA HPWB HPWC Prostate BPH Uni-ZAP XR LP013 HPWD HPWE HPIA HPIB HPIC LNCAP prostate cell line Uni-ZAP XR LP013 HPJA HPJB HPJC PC3 Prostate cell line Uni-ZAP XR LP013 HBTA Bone Marrow Stroma, Uni-ZAP XR LP013 TNF&LPS ind HMCF HMCG HMCH HMCI Macrophage-oxLDL; re- Uni-ZAP XR LP013 HMCJ excision HAGG HAGH HAGI Human Amygdala;re-excision Uni-ZAP XR LP013 HACA H. Adipose Tissue Uni-ZAP XR LP013 HKFB K562 + PMA (36 hrs), re- ZAP Express LP013 excision HCWT HCWU HCWV CD34 positive cells (cord ZAP Express LP013 blood), re-ex HBWA Whole brain ZAP Express LP013 HBXA HBXB HBXC HBXD Human Whole Brain #2 - ZAP Express LP013 Oligo dT > 1.5Kb HAVM Temporal cortex-Alzheizmer pT-Adv LP014 HAVT Hippocampus, Alzheimer pT-Adv LP014 Subtracted HHAS CHME Cell Line Uni-ZAP XR LP014 HAJR Larynx normal pSport 1 LP014 HWLE HWLF HWLG Colon Normal pSport 1 LP014 HWLH HCRM HCRN HCRO Colon Carcinoma pSport 1 LP014 HWLI HWLJ HWLK Colon Normal pSport 1 LP014 HWLQ HWLR HWLS Colon Tumor pSport 1 LP014 HWLT HBFM Gastrocnemius Muscle pSport 1 LP014 HBOD HBOE Quadriceps Muscle pSport 1 LP014 HBKD HBKE Soleus Muscle pSport 1 LP014 HCCM Pancreatic Langerhans pSport 1 LP014 HWGA Larynx carcinoma pSport 1 LP014 HWGM HWGN Larynx carcinoma pSport 1 LP014 HWLA HWLB HWLC Normal colon pSport 1 LP014 HWLM HWLN Colon Tumor pSport 1 LP014 HVAM HVAN HVAO Pancreas Tumor pSport 1 LP014 HWGQ Larynx carcinoma pSport 1 LP014 HAQM HAQN Salivary Gland pSport 1 LP014 HASM Stomach; normal pSport 1 LP014 HBCM Uterus; normal pSport 1 LP014 HCDM Testis; normal pSport 1 LP014 HDJM Brain; normal pSport 1 LP014 HEFM Adrenal Gland, normal pSport 1 LP014 HBAA Rectum normal pSport 1 LP014 HFDM Rectum tumour pSport 1 LP014 HGAM Colon, normal pSport 1 LP014 HHMM Colon, tumour pSport 1 LP014 HCLB HCLC Human Lung Cancer Lambda Zap II LP015 HRLA L1 Cell line ZAP Express LP015 HHAM Hypothalamus, Alzheimer's pCMVSport 3.0 LP015 HKBA Ku 812F Basophils Line pSport 1 LP015 HS2S Saos2, Dexamethosome pSport 1 LP016 Treated HASA Lung Carcinoma A549 pSport 1 LP016 TNFalpha activated HTFM TF-1 Cell Line GM-CSF pSport 1 LP016 Treated HYAS Thyroid Tumour pSport 1 LP016 HUTS Larynx Normal pSport 1 LP016 HXOA Larynx Tumor pSport 1 LP016 HEAH Ea.hy.926 cell line pSport 1 LP016 HINA Adenocarcinoma Human pSport 1 LP016 HRMA Lung Mesothelium pSport 1 LP016 HLCL Human Pre-Differentiated Uni-Zap XR LP017 Adipocytes HS2A Saos2 Cells pSport 1 LP020 HS2I Saos2 Cells; Vitamin D3 pSport 1 LP020 Treated HUCM CHME Cell Line, untreated pSport 1 LP020 HEPN Aryepiglottis Normal pSport 1 LP020 HPSN Sinus Piniformis Tumour pSport 1 LP020 HNSA Stomach Normal pSport 1 LP020 HNSM Stomach Tumour pSport 1 LP020 HNLA Liver Normal Met5No pSport 1 LP020 HUTA Liver Tumour Met 5 Tu pSport 1 LP020 HOCN Colon Normal pSport 1 LP020 HOCT Colon Tumor pSport 1 LP020 HTNT Tongue Tumour pSport 1 LP020 HLXN Larynx Normal pSport 1 LP020 HLXT Larynx Tumour pSport 1 LP020 HTYN Thymus pSport 1 LP020 HPLN Placenta pSport 1 LP020 HTNG Tongue Normal pSport 1 LP020 HZAA Thyroid Normal (SDCA2 No) pSport 1 LP020 HWES Thyroid Thyroiditis pSport 1 LP020 HFHD FICOLL ™ed Human Stromal pTrip1Ex2 LP021 Cells, 5Fu treated HFHM,HFHN FICOLL ™ed Human Stromal pTrip1Ex2 LP021 Cells, Untreated HPCI Hep G2 Cells, lambda library lambda Zap-CMV LP021 XR HBCA, HBCB, HBCC H. Lymph node breast Cancer Uni-ZAP XR LP021 HCOK Chondrocytes pSPORT1 LP022 HDCA, HDCB, HDCC Dendritic Cells From CD34 pSPORT1 LP022 Cells HDMA, HDMB CD40 activated monocyte pSPORT1 LP022 dendritic cells HDDM, HDDN, HDDO LPS activated derived pSPORT1 LP022 dendritic cells HPCR Hep G2 Cells, PCR library lambda Zap-CMV LP022 XR HAAA, HAAB, HAAC Lung, Cancer (4005313A3): pSPORT1 LP022 Invasive Poorly Differentiated Lung Adenocarcinoma HIPA, HIPB, HIPC Lung, Cancer (4005163 B7): pSPORT1 LP022 Invasive, Poorly Diff. Adenocarcinoma, Metastatic HOOH, HOOI Ovary, Cancer: (4004562 B6) pSPORT1 LP022 Papillary Serous Cystic Neoplasm, Low Malignant Pot HIDA Lung, Normal: (4005313 B1) pSPORT1 LP022 HUJA, HUJB, HUJC, HUJD, B-Cells pCMVSport 3.0 LP022 HUJE HNOA, HNOB, HNOC, HNOD Ovary, Normal: (9805C040R) pSPORT1 LP022 HNLM Lung, Normal: (4005313 B1) pSPORT1 LP022 HSCL Stromal Cells pSPORT1 LP022 HAAX Lung, Cancer: (4005313 A3) pSPORT1 LP022 Invasive Poorly-differentiated Metastatic lung adenocarcinoma HUUA,HUUB,HUUC,HUUD B-cells (unstimulated) pTriplEx2 LP022 HWWA, HWWB, HWWC, B-cells (stimulated) pSPORT1 LP022 HWWD, HWWE, HWWF, HWWG HCCC Colon, Cancer: (9808C064R) pCMVSport 3.0 LP023 HPDO HPDP HPDQ HPDR Ovary, Cancer (9809C332): pSport 1 LP023 HPD Poorly differentiated adenocarcinoma HPCO HPCP HPCQ HPCT Ovary, Cancer (15395A1F): pSport 1 LP023 Grade II Papillary Carcinoma HOCM HOCO HOCP HOCQ Ovary, Cancer: (15799A1F) pSport 1 LP023 Poorly differentiated carcinoma HCBM HCBN HCBO Breast, Cancer: (4004943 A5) pSport 1 LP023 HNBT HNBU HNBV Breast, Normal: (4005522B2) pSport 1 LP023 HBCP HBCQ Breast, Cancer: (4005522 A2) pSport 1 LP023 HBCJ Breast, Cancer: (9806C012R) pSport 1 LP023 HSAM HSAN Stromal cells 3.88 pSport 1 LP023 HVCA HVCB HVCC HVCD Ovary, Cancer: (4004332 A2) pSport 1 LP023 HSCK HSEN HSEO Stromal cells (HBM3.18) pSport 1 LP023 HSCP HSCQ stromal cell clone 2.5 pSport 1 LP023 HUXA Breast Cancer: (4005385 A2) pSport 1 LP023 HCOM HCON HCOO HCOP Ovary, Cancer (4004650 A3): pSport 1 LP023 HCOQ Well-Differentiated Micropapillary Serous Carcinoma HBNM Breast, Cancer: (9802C020E) pSport 1 LP023 HVVA HVVB HVVC HVVD Human Bone Marrow, treated pSport 1 LP023 HVVE

Two nonlimiting examples are provided below for isolating a particular clone from the deposited sample of plasmid cDNAs cited for that clone in Table 1A. First, a plasmid is directly isolated by screening the clones using a polynucleotide probe corresponding to the nucleotide sequence of SEQ ID NO:X.

Particularly, a specific polynucleotide with 30-40 nucleotides is synthesized using an Applied Biosystems DNA synthesizer according to the sequence reported. The oligonucleotide is labeled, for instance, with ³²P-γ-ATP using T4 polynucleotide kinase and purified according to routine methods. (E.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982)). The plasmid mixture is transformed into a suitable host, as indicated above (such as XL-1 Blue (STRATAGENE™)) using techniques known to those of skill in the art, such as those provided by the vector supplier or in related publications or patents cited above. The transformants are plated on 1.5% agar plates (containing the appropriate selection agent, e.g., ampicillin) to a density of about 150 transformants (colonies) per plate. These plates are screened using Nylon membranes according to routine methods for bacterial colony screening (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages 1.93 to 1.104), or other techniques known to those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both ends of the nucleotide sequence of SEQ ID NO:X are synthesized and used to amplify the desired cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 μl of reaction mixture with 0.5 μg of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94° C. for 1 min; annealing at 55° C. for 1 min; elongation at 72° C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product.

Several methods are available for the identification of the 5′ or 3′ non-coding portions of a gene which may not be present in the deposited clone. These methods include but are not limited to, filter probing, clone enrichment using specific probes, and protocols similar or identical to 5′ and 3′ “RACE” protocols which are well known in the art. For instance, a method similar to 5′ RACE is available for generating the missing 5′ end of a desired full-length transcript. (Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993)).

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of a population of RNA presumably containing full-length gene RNA transcripts. A primer set containing a primer specific to the ligated RNA oligonucleotide and a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5′ portion of the desired full-length gene. This amplified product may then be sequenced and used to generate the full length gene.

This above method starts with total RNA isolated from the desired source, although poly-A+ RNA can be used. The RNA preparation can then be treated with phosphatase if necessary to eliminate 5′ phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase should then be inactivated and the RNA treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5′ ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the 5′ end of the cap cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strand cDNA synthesis using a gene specific oligonucleotide. The first strand synthesis reaction is used as a template for PCR amplification of the desired 5′ end using a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5′ end sequence belongs to the desired gene.

Example 2 Isolation of Genomic Clones Corresponding to a Polynucleotide

A human genomic P1 library (Genomic Systems, Inc.) is screened by PCR using primers selected for the sequence corresponding to SEQ ID NO:X according to the method described in Example 1. (See also, Sambrook.)

Example 3 Tissue Specific Expression Analysis

Tissue distribution of mRNA expression of polynucleotides of the present invention is determined using protocols for Northern blot analysis, described by, among others, Sambrook et al. For example, a cDNA probe produced by the method described in Example 1 is labeled with P³² using the REDIPRIME™ DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using CHROMA SPN-100™ column (CLONTECH™ Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to examine various human tissues for mRNA expression.

Multiple Tissue Northern (MTN) blots containing various human tissues (H) or human immune system tissues (IM) (CLONTECH™) are examined with the labeled probe using EXPRESSHYB™ hybridization solution (CLONTECH™) according to manufacturer's protocol number PT1190-1. Following hybridization and washing, the blots are mounted and exposed to film at −70° C. overnight, and the films developed according to standard procedures.

The Human Genome Sciences, Inc. (HGS) database is derived from sequencing tissue and/or disease specific cDNA libraries. Libraries generated from a particular tissue are selected and the specific tissue expression pattern of EST groups or assembled contigs within these libraries is determined by comparison of the expression patterns of those groups or contigs within the entire database. ESTs and assembled contigs which show tissue specific expression are selected.

The original clone from which the specific EST sequence was generated, or in the case of an assembled contig, the clone from which the 5′ most EST sequence was generated, is obtained from the catalogued library of clones and the insert amplified by PCR using methods known in the art. The PCR product is denatured and then transferred in 96 or 384 well format to a nylon membrane (Schleicher and Scheull) generating an array filter of tissue specific clones. Housekeeping genes, maize genes, and known tissue specific genes are included on the filters. These targets can be used in signal normalization and to validate assay sensitivity. Additional targets are included to monitor probe length and specificity of hybridization.

Radioactively labeled hybridization probes are generated by first strand cDNA synthesis per the manufacturer's instructions (LIFE TECHNOLOGIES™) from mRNA/RNA samples prepared from the specific tissue being analyzed (e.g., prostate, prostate cancer, ovarian, ovarian cancer, etc.). The hybridization probes are purified by gel exclusion chromatography, quantitated, and hybridized with the array filters in hybridization bottles at 65° C. overnight. The filters are washed under stringent conditions and signals are captured using a Fuji phosphorimager.

Data is extracted using AIS software and following background subtraction, signal normalization is performed. This includes a normalization of filter-wide expression levels between different experimental runs. Genes that are differentially expressed in the tissue of interest are identified.

Example 4 Chromosomal Mapping of the Polynucleotides

An oligonucleotide primer set is designed according to the sequence at the 5′ end of SEQ ID NO:X. This primer preferably spans about 100 nucleotides. This primer set is then used in a polymerase chain reaction under the following set of conditions: 30 seconds, 95° C.; 1 minute, 56° C.; 1 minute, 70° C. This cycle is repeated 32 times followed by one 5 minute cycle at 70° C. Human, mouse, and hamster DNA is used as template in addition to a somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc). The reactions are analyzed on either 8% polyacrylamide gels or 3.5% agarose gels. Chromosome mapping is determined by the presence of an approximately 100 by PCR fragment in the particular somatic cell hybrid.

Example 5 Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention is amplified using PCR oligonucleotide primers corresponding to the 5′ and 3′ ends of the DNA sequence, as outlined in Example 1, to synthesize insertion fragments. The primers used to amplify the cDNA insert should preferably contain restriction sites, such as BamHI and XbaI, at the 5′ end of the primers in order to clone the amplified product into the expression vector. For example, BamHI and XbaI correspond to the restriction enzyme sites on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodes antibiotic resistance (Amp^(r)), a bacterial origin of replication (ori), an IPTG-regulatable promoter/operator (P/O), a ribosome binding site (RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplified fragment is ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture is then used to transform the E. coli strain M15/rep4 (Qiagen, Inc.) which contains multiple copies of the plasmid pREP4, which expresses the lad repressor and also confers kanamycin resistance (Kan^(r)). Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both Amp (100 μg/ml) and Kan (25 μg/ml). The 0/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lad repressor, clearing the P/O leading to increased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 min at 6000×g). The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at 4° C. The cell debris is removed by centrifugation, and the supernatant containing the polypeptide is loaded onto a nickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc., supra). Proteins with a 6×His tag bind to the Ni-NTA resin with high affinity and can be purified in a simple one-step procedure (for details see: The QIAexpressionist (1995) QIAGEN, Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl, pH 8. The column is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it against phosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus 200 mM NaCl. Alternatively, the protein can be successfully refolded while immobilized on the Ni-NTA column. The recommended conditions are as follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. The renaturation should be performed over a period of 1.5 hours or more. After renaturation the proteins are eluted by the addition of 250 mM immidazole. Immidazole is removed by a final dialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified protein is stored at 4° C. or frozen at −80° C.

In addition to the above expression vector, the present invention further includes an expression vector, called pHE4a (ATCC™ Accession Number 209645, deposited on Feb. 25, 1998) which contains phage operator and promoter elements operatively linked to a polynucleotide of the present invention, called pHE4a. (ATCC™ Accession Number 209645, deposited on Feb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferase gene as a selection marker, 2) an E. coli origin of replication, 3) a T5 phage promoter sequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence, and 6) the lactose operon repressor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). The promoter and operator sequences are made synthetically.

DNA can be inserted into the pHE4a by restricting the vector with NdeI and XbaI, BamHI, XhoI, or Asp718, running the restricted product on a gel, and isolating the larger fragment (the stuffer fragment should be about 310 base pairs). The DNA insert is generated according to the PCR protocol described in Example 1, using PCR primers having restriction sites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.

The engineered vector could easily be substituted in the above protocol to express protein in a bacterial system.

Example 6 Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptide expressed in E. coli when it is present in the form of inclusion bodies. Unless otherwise specified, all of the following steps are conducted at 4-10° C.

Upon completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10° C. and the cells harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste, by weight, is suspended in a buffer solution containing 100 mM Tris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneous suspension using a high shear mixer.

The cells are then lysed by passing the solution through a microfluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The homogenate is then mixed with NaCl solution to a final concentration of 0.5 M NaCl, followed by centrifugation at 7000×g for 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mM Tris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 M guanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×g centrifugation for 15 min., the pellet is discarded and the polypeptide containing supernatant is incubated at 4° C. overnight to allow further GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insoluble particles, the GuHCl solubilized protein is refolded by quickly mixing the GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH 4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded diluted protein solution is kept at 4° C. without mixing for 12 hours prior to further purification steps.

To clarify the refolded polypeptide solution, a previously prepared tangential filtration unit equipped with 0.16 μm membrane filter with appropriate surface area (e.g., Filtron), equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filtered sample is loaded onto a cation exchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column is washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. The absorbance at 280 nm of the effluent is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4 volumes of water. The diluted sample is then loaded onto a previously prepared set of tandem columns of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchange resins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10 column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractions are collected under constant A₂₈₀ monitoring of the effluent. Fractions containing the polypeptide (determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity after the above refolding and purification steps. No major contaminant bands should be observed from Commassie blue stained 16% SDS-PAGE gel when 5 μg of purified protein is loaded. The purified protein can also be tested for endotoxin/LPS contamination, and typically the LPS content is less than 0.1 ng/ml according to LAL assays.

Example 7 Cloning and Expression of a Polypeptide in a Baculovirus Expression System

In this example, the plasmid shuttle vector pA2 is used to insert a polynucleotide into a baculovirus to express a polypeptide. This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites such as BamHI, Xba I and Asp718. The polyadenylation site of the simian virus 40 (“SV40”) is used for efficient polyadenylation. For easy selection of recombinant virus, the plasmid contains the beta-galactosidase gene from E. coli under control of a weak Drosophila promoter in the same orientation, followed by the polyadenylation signal of the polyhedrin gene. The inserted genes are flanked on both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate a viable virus that express the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above, such as pAc373, pVL941, and pAcIM1, as one skilled in the art would readily appreciate, as long as the construct provides appropriately located signals for transcription, translation, secretion and the like, including a signal peptide and an in-frame AUG as required. Such vectors are described, for instance, in Luckow et al., Virology 170:31-39 (1989).

Specifically, the cDNA sequence contained in the deposited clone, including the AUG initiation codon, is amplified using the PCR protocol described in Example 1. If a naturally occurring signal sequence is used to produce the polypeptide of the present invention, the pA2 vector does not need a second signal peptide. Alternatively, the vector can be modified (pA2 GP) to include a baculovirus leader sequence, using the standard methods described in Summers et al., “A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures,” Texas Agricultural Experimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“GENECLEAN™,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes and optionally, can be dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art. The DNA is then isolated from a 1% agarose gel using a commercially available kit (“GENECLEAN™” BIO 101 Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together with T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such as XL-1 Blue (STRATAGENE™ Cloning Systems, La Jolla, Calif.) cells are transformed with the ligation mixture and spread on culture plates. Bacteria containing the plasmid are identified by digesting DNA from individual colonies and analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing.

Five μg of a plasmid containing the polynucleotide is co-transfected with 1.0 μg of a commercially available linearized baculovirus DNA (“BACULOGOLD™ baculovirus DNA, Pharmingen, San Diego, Calif.), using the lipofection method described by Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). One μg of BACULOGOLD™ virus DNA and 5 μg of the plasmid are mixed in a sterile well of a microtiter plate containing 50 μl of serum-free Grace's medium (LIFE TECHNOLOGIES™ Inc., Gaithersburg, Md.). Afterwards, 10 μl LIPOFECTIN™ plus 90 μl Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC™ CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is then incubated for 5 hours at 27° C. The transfection solution is then removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added. Cultivation is then continued at 27° C. for four days.

After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, supra. An agarose gel with “Blue Gal” (LIFE TECHNOLOGIES™ Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a “plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by LIFE TECHNOLOGIES™ Inc., Gaithersburg, page 9-10.) After appropriate incubation, blue stained plaques are picked with the tip of a micropipettor (e.g., Eppendorf). The agar containing the recombinant viruses is then resuspended in a microcentrifuge tube containing 200 μl of Grace's medium and the suspension containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4° C.

To verify the expression of the polypeptide, Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus containing the polynucleotide at a multiplicity of infection (“MOI”) of about 2. If radiolabeled proteins are desired, 6 hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from LIFE TECHNOLOGIES™ Inc., Rockville, Md.). After 42 hours, 5 μCi of ³⁵5-methionine and 5 μCi ³⁵5-cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then are harvested by centrifugation. The proteins in the supernatant as well as the intracellular proteins are analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus of purified protein may be used to determine the amino terminal sequence of the produced protein.

Example 8 Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammalian cell. A typical mammalian expression vector contains a promoter element, which mediates the initiation of transcription of mRNA, a protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription is achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (PHARMACIA™, Uppsala, Sweden), pRSVcat (ATCC™ 37152), pSV2dhfr (ATCC™ 37146), pBC12MI (ATCC™ 67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells that could be used include, human Hela, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell lines containing the polynucleotide integrated into a chromosome. The co-transfection with a selectable marker such as DHFR, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful in developing cell lines that carry several hundred or even several thousand copies of the gene of interest. (See, e.g., Alt, F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A., Biotechnology 9:64-68 (1991)). Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of proteins.

Derivatives of the plasmid pSV2-dhfr (ATCC™ Accession No. 37146), the expression vectors pC4 (ATCC™ Accession No. 209646) and pC6 (ATCC™ Accession No. 209647) contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning of the gene of interest. The vectors also contain the 3′ intron, the polyadenylation and termination signal of the rat preproinsulin gene, and the mouse DHFR gene under control of the SV40 early promoter.

Specifically, the plasmid pC6, for example, is digested with appropriate restriction enzymes and then dephosphorylated using calf intestinal phosphates by procedures known in the art. The vector is then isolated from a 1% agarose gel.

A polynucleotide of the present invention is amplified according to the protocol outlined in Example 1. If a naturally occurring signal sequence is used to produce the polypeptide of the present invention, the vector does not need a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., International Publication No. WO 96/34891.)

The amplified fragment is isolated from a 1% agarose gel using a commercially available kit (“GENECLEAN™,” BIO 101 Inc., La Jolla, Calif.). The fragment then is digested with appropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzyme and purified on a 1% agarose gel. The isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC6 using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used for transfection. Five μg of the expression plasmid pC6 or pC4 is cotransfected with 0.5 μg of the plasmid pSVneo using LIPOFECTIN™ (Felgner et al., supra). The plasmid pSV2-neo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained which grow at a concentration of 100-200 μM. Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reversed phase HPLC analysis.

Example 9 Protein Fusions

The polypeptides of the present invention are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of the present polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example 5; see also EPA 394,827; Traunecker, et al., Nature 331:84-86 (1988)). Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclear localization signals fused to the polypeptides of the present invention can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule, or the protocol described in Example 5.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector.

For example, if pC4 (ATCC™ Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3′ BamHI site should be destroyed. Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and a polynucleotide of the present invention, isolated by the PCR protocol described in Example 1, is ligated into this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.

If the naturally occurring signal sequence is used to produce the polypeptide of the present invention, pC4 does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. (See, e.g., International Publication No. WO 96/34891.)

(SEQ ID NO: 236) Human IgG Fc region: GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACC TGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCA TGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCC TGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAG CCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGC ACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCC AACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGT GTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGC CTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC CGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG GGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 10 Production of an Antibody from a Polypeptide a) Hybridoma Technology

The antibodies of the present invention can be prepared by a variety of methods. (See, Current Protocols, Chapter 2.) As one example of such methods, cells expressing a polypeptide of the present invention are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of a polypeptide of the present invention is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

Monoclonal antibodies specific for a polypeptide of the present invention are prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681 (1981)). In general, an animal (preferably a mouse) is immunized with a polypeptide of the present invention or, more preferably, with a secreted polypeptide-expressing cell. Such polypeptide-expressing cells are cultured in any suitable tissue culture medium, preferably in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC™. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide of the present invention.

Alternatively, additional antibodies capable of binding to a polypeptide of the present invention can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody that binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the polypeptide-specific antibody can be blocked by said polypeptide. Such antibodies comprise anti-idiotypic antibodies to the polypeptide-specific antibody and are used to immunize an animal to induce formation of further polypeptide-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). Alternatively, secreted protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.

For in vivo use of antibodies in humans, an antibody is “humanized”. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., International Publication No. WO 8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985)).

b) Isolation of Antibody Fragments Directed Against a Polypeptide of the Present Invention from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructed into a library of antibody fragments which contain reactivities against a polypeptide of the present invention to which the donor may or may not have been exposed (see e.g., U.S. Pat. No. 5,885,793 incorporated herein by reference in its entirety).

Rescue of the Library.

A library of scFvs is constructed from the RNA of human PBLs as described in International Publication No. WO 92/01047. To rescue phage displaying antibody fragments, approximately 10⁹ E. coli harboring the phagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and 100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking Five ml of this culture is used to inoculate 50 ml of 2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, see International Publication No. WO 92/01047) are added and the culture incubated at 37° C. for 45 minutes without shaking and then at 37° C. for 45 minutes with shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillin and 50 μg/ml kanamycin and grown overnight. Phage are prepared as described in International Publication No. WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious M13 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37° C. without shaking and then for a further hour at 37° C. with shaking Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min), resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25 μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C. Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS and passed through a 0.45 μm filter (Minisart NML; Sartorius) to give a final concentration of approximately 10¹³ transducing units/ml (ampicillin-resistant clones).

Panning of the Library.

Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C. and then washed 3 times in PBS. Approximately 10¹³ TU of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS. Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37° C. The E. coli are then plated on TYE plates containing 1% glucose and 100 μg/ml ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection. This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders.

Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks, et al., 1991) from single colonies for assay. ELISAs are performed with microtitre plates coated with either 10 pg/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (see, e.g., International Publication No. WO 92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.

Example 11 Method of Determining Alterations in a Gene Corresponding to a Polynucleotide

RNA from entire families or individual patients presenting with a phenotype of interest (such as a disease or disorder, e.g., an immune, cardiovascular, cancer, or other proliferative disease or disorder) is isolated. cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook.) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO:X; and/or the nucleotide sequence of the cDNA contained in ATCC™ Deposit No:Z. Suggested PCR conditions consist of 35 cycles at 95° C. for 30 seconds; 60-120 seconds at 52-58° C.; and 60-120 seconds at 70° C., using buffer solutions described in Sidransky et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase (Epicentre Technologies). The intron-exon boundaries of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations are then cloned and sequenced to validate the results of the direct sequencing.

PCR products are cloned into T-tailed vectors as described in Holton et al., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determining alterations in a gene corresponding to a polynucleotide. Genomic clones isolated according to Example 2 are nick-translated with digoxigenindeoxy-uridine 5′-triphosphate (BOEHRINGER™ Manheim), and FISH performed as described in Johnson et al., Methods Cell Biol. 35:73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. (Johnson et al., Genet. Anal. Tech. Appl., 8:75 (1991)). Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease.

Example 12 Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample

A polypeptide of the present invention can be detected in a biological sample, and if an increased or decreased level of the polypeptide is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 μg/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described in Example 10. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbound polypeptide.

Next, 50 μl of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbound conjugate.

Add 75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate 1 hour at room temperature. Measure the reaction by a microtiter plate reader. Prepare a standard curve, using serial dilutions of a control sample, and plot polypeptide concentration on the X-axis (log scale) and fluorescence or absorbance of the Y-axis (linear scale). Interpolate the concentration of the polypeptide in the sample using the standard curve.

Example 13 Formulation

The invention also provides methods of preventing, treating and/or ameliorating a disease or disorder (such as, for example, any one or more of the diseases or disorders disclosed herein, e.g., an immune, cardiovascular, cancer, or other proliferative disease or disorder) by administration to a subject of an effective amount of a Therapeutic. By therapeutic is meant polynucleotides or polypeptides of the invention (including fragments and variants), agonists or antagonists thereof, and/or antibodies thereto, in combination with a pharmaceutically acceptable carrier type (e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the Therapeutic alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount of the Therapeutic administered parenterally per dose will be in the range of about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the Therapeutic is typically administered at a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect.

Therapeutics can be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Therapeutics of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release Therapeutics include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (Langer et al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988).

In a preferred embodiment, polypeptide, polynucleotide, and antibody compositions of the invention are formulated in a biodegradable, polymeric drug delivery system, for example as described in U.S. Pat. Nos. 4,938,763; 5,278,201; 5,278,202; 5,324,519; 5,340,849; and 5,487,897 and in International Publication Numbers WO01/35929, WO00/24374, and WO00/06117 which are hereby incorporated by reference in their entirety. In specific preferred embodiments the polypeptide, polynucleotide, and antibody compositions of the invention are formulated using the ATRIGEL® Biodegradable System of Atrix Laboratories, Inc. (Fort Collins, Colo.).

Examples of biodegradable polymers which can be used in the formulation of polypeptide, polynucleotide, and antibody compositions, include but are not limited to, polylactides, polyglycolides, polycaprolactones, polyanhydrides, polyamides, polyurethanes, polyesteramides, polyorthoesters, polydioxanones, polyacetals, polyketals, polycarbonates, polyorthocarbonates, polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates, polyalkylene oxalates, polyalkylene succinates, poly(malic acid), poly(amino acids), poly(methyl vinyl ether), poly(maleic anhydride), polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, chitin, chitosan, and copolymers, terpolymers, or combinations or mixtures of the above materials. The preferred polymers are those that have a lower degree of crystallization and are more hydrophobic. These polymers and copolymers are more soluble in the biocompatible solvents than the highly crystalline polymers such as polyglycolide and chitin which also have a high degree of hydrogen-bonding. Preferred materials with the desired solubility parameters are the polylactides, polycaprolactones, and copolymers of these with glycolide in which there are more amorphous regions to enhance solubility. In specific preferred embodiments, the biodegradable polymers which can be used in the formulation of polypeptide, polynucleotide, and antibody compositions are poly(lactide-co-glycolides). Polymer properties such as molecular weight, hydrophobicity, and lactide/glycolide ratio may be modified to obtain the desired polypeptide, polynucleotide, or antibody release profile (See, e.g., Ravivarapu et al., Journal of Pharmaceutical Sciences 89:732-741 (2000), which is hereby incorporated by reference in its entirety).

It is also preferred that the solvent for the biodegradable polymer be non-toxic, water miscible, and otherwise biocompatible. Examples of such solvents include, but are not limited to, N-methyl-2-pyrrolidone, 2-pyrrolidone, C2 to C6 alkanols, C1 to C15 alchohols, dils, triols, and tetraols such as ethanol, glycerine propylene glycol, butanol; C3 to C15 alkyl ketones such as acetone, diethyl ketone and methyl ethyl ketone; C3 to C15 esters such as methyl acetate, ethyl acetate, ethyl lactate; alkyl ketones such as methyl ethyl ketone, C1 to C15 amides such as dimethylformamide, dimethylacetamide and caprolactam; C3 to C20 ethers such as tetrahydrofuran, or solketal; tweens, triacetin, propylene carbonate, decylmethylsulfoxide, dimethyl sulfoxide, oleic acid, 1-dodecylazacycloheptan-2-one, Other preferred solvents are benzyl alchohol, benzyl benzoate, dipropylene glycol, tributyrin, ethyl oleate, glycerin, glycofural, isopropyl myristate, isopropyl palmitate, oleic acid, polyethylene glycol, propylene carbonate, and triethyl citrate. The most preferred solvents are N-methyl-2-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, triacetin, and propylene carbonate because of the solvating ability and their compatibility.

Additionally, formulations comprising polypeptide, polynucleotide, and antibody compositions and a biodegradable polymer may also include release-rate modification agents and/or pore-forming agents. Examples of release-rate modification agents include, but are not limited to, fatty acids, triglycerides, other like hydrophobic compounds, organic solvents, plasticizing compounds and hydrophilic compounds. Suitable release rate modification agents include, for example, esters of mono-, di-, and tricarboxylic acids, such as 2-ethoxyethyl acetate, methyl acetate, ethyl acetate, diethyl phthalate, dimethyl phthalate, dibutyl phthalate, dimethyl adipate, dimethyl succinate, dimethyl oxalate, dimethyl citrate, triethyl citrate, acetyl tributyl citrate, acetyl triethyl citrate, glycerol triacetate, di(n-butyl) sebecate, and the like; polyhydroxy alcohols, such as propylene glycol, polyethylene glycol, glycerin, sorbitol, and the like; fatty acids; triesters of glycerol, such as triglycerides, epoxidized soybean oil, and other epoxidized vegetable oils; sterols, such as cholesterol; alcohols, such as C.sub.6-C.sub.12 alkanols, 2-ethoxyethanol. The release rate modification agent may be used singly or in combination with other such agents. Suitable combinations of release rate modification agents include, but are not limited to, glycerin/propylene glycol, sorbitol/glycerine, ethylene oxide/propylene oxide, butylene glycol/adipic acid, and the like. Preferred release rate modification agents include, but are not limited to, dimethyl citrate, triethyl citrate, ethyl heptanoate, glycerin, and hexanediol. Suitable pore-forming agents that may be used in the polymer composition include, but are not limited to, sugars such as sucrose and dextrose, salts such as sodium chloride and sodium carbonate, polymers such as hydroxylpropylcellulose, carboxymethylcellulose, polyethylene glycol, and polyvinylpyrrolidone. Solid crystals that will provide a defined pore size, such as salt or sugar, are preferred.

In specific preferred embodiments the polypeptide, polynucleotide, and antibody compositions of the invention are formulated using the BEMA™ BioErodible Mucoadhesive System, MCA™ MucoCutaneous Absorption System, SMP™ Solvent MicroParticle System, or BCP™ BioCompatible Polymer System of Atrix Laboratories, Inc. (Fort Collins, Colo.).

Sustained-release Therapeutics also include liposomally entrapped Therapeutics of the invention (see generally, Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)). Liposomes containing the Therapeutic are prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal Therapeutic.

In yet an additional embodiment, the Therapeutics of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, i.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeutic uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutics generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous Therapeutic solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized Therapeutic using bacteriostatic Water-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the Therapeutics of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the Therapeutics may be employed in conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or in combination with adjuvants. Adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (GENENTECH™, Inc.), BCG (e.g., THERACYS®), MPL and nonviable prepartions of Corynebacterium parvum. In a specific embodiment, Therapeutics of the invention are administered in combination with alum. In another specific embodiment, Therapeutics of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the Therapeutics of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the Therapeutics of the invention include, but are not limited to, vaccines directed toward protection against MMR (measles, mumps, rubella), polio, varicella, tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B, whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus, cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies, typhoid fever, and pertussis. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

The Therapeutics of the invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered in combination with the Therapeutics of the invention, include but are not limited to, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines, growth factors, and/or therapeutic treatments described below. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration “in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.

In one embodiment, the Therapeutics of the invention are administered in combination with an anticoagulant. Anticoagulants that may be administered with the compositions of the invention include, but are not limited to, heparin, low molecular weight heparin, warfarin sodium (e.g., COUMADIN®), dicumarol, 4-hydroxycoumarin, anisindione (e.g., MIRADON™), acenocoumarol (e.g., nicoumalone, SINTHROME™), indan-1,3-dione, phenprocoumon (e.g., MARCUMAR™), ethyl biscoumacetate (e.g., TROMEXAN™), and aspirin. In a specific embodiment, compositions of the invention are administered in combination with heparin and/or warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin. In another specific embodiment, compositions of the invention are administered in combination with warfarin and aspirin. In another specific embodiment, compositions of the invention are administered in combination with heparin. In another specific embodiment, compositions of the invention are administered in combination with heparin and aspirin.

In another embodiment, the Therapeutics of the invention are administered in combination with thrombolytic drugs. Thrombolytic drugs that may be administered with the compositions of the invention include, but are not limited to, plasminogen, lys-plasminogen, alpha2-antiplasmin, streptokinae (e.g., KABIKINASE™), antiresplace (e.g., EMINASE™), tissue plasminogen activator (t-PA, altevase, ACTIVASE™), urokinase (e.g., ABBOKINASE™), sauruplase, (Prourokinase, single chain urokinase), and aminocaproic acid (e.g., AMICAR™). In a specific embodiment, compositions of the invention are administered in combination with tissue plasminogen activator and aspirin.

In another embodiment, the Therapeutics of the invention are administered in combination with antiplatelet drugs. Antiplatelet drugs that may be administered with the compositions of the invention include, but are not limited to, aspirin, dipyridamole (e.g., PERSANTINE™), and ticlopidine (e.g., TICLID™).

In specific embodiments, the use of anti-coagulants, thrombolytic and/or antiplatelet drugs in combination with Therapeutics of the invention is contemplated for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of thrombosis, arterial thrombosis, venous thrombosis, thromboembolism, pulmonary embolism, atherosclerosis, myocardial infarction, transient ischemic attack, unstable angina. In specific embodiments, the use of anticoagulants, thrombolytic drugs and/or antiplatelet drugs in combination with Therapeutics of the invention is contemplated for the prevention of occulsion of saphenous grafts, for reducing the risk of periprocedural thrombosis as might accompany angioplasty procedures, for reducing the risk of stroke in patients with atrial fibrillation including nonrheumatic atrial fibrillation, for reducing the risk of embolism associated with mechanical heart valves and or mitral valves disease. Other uses for the therapeutics of the invention, alone or in combination with antiplatelet, anticoagulant, and/or thrombolytic drugs, include, but are not limited to, the prevention of occlusions in extracorporeal devices (e.g., intravascular canulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines).

Therapeutics of the invention may also be administered in combination with additional cardiovascular agents, such as, for example, beta-adrenergic blockers, calcium channel blockers, ACE inhibitors, angiotensin II blockers, alpha adrenergic blockers, hypotensive agents, antilipemic agents, and vasodilating agents.

Non-limiting examples of beta-adrenergic blockers includes TENORMIN™ (atenolol), BREVIBLOC™ (esmolol), NORMODYNE™ (labetalol), TRANDATE™, LOPRESSOR™ (metoprolol), INDERAL™ (propranolol), and BETApp96™ (sotalol). Calcium channel blockers includes, for example, NORVASC™ (amlodipine), CARDIZEM™ (diltiazem), PLENDIL™ (felodipine), DYNACRIC™ (isradipine), CARDENE™ (nicardipine), ADALAT™ (nifedipine), and CALAN™ (verapamil). ACE inhibitors includes, for example, LOTENSIN™ (benazepril), CAPOTEN™ (captopril), VASOTEC™ (enalapril), MONOPRIL™ (fosinopril), PRINIVIL™ (lisinopril), ACCUPRIL™ (quinapril), and ALTACE™ (ramipril). Non-limiting examples of angiotensin II blockers includes AVAPRO™ (irbesartan), COZAAR™ (losartan), and DIOVAN™ (valsartan). Alpha adrenergic blockers include, for example, CARDURA™ (doxazosin), MINIPRESS™ (prazosin), FLOMAX™ (tamsulosin), and terazosin. Hypotensive agents include, for example, CATAPRES™ (clonidine), APRESOLINE™ (hydralazine), ALDOMET™ (methyldopa), LONITEN™ (minoxidil), NIPRIDE™ (nitroprusside) and reserpine. Antilipemic agents include, for example, LIPITOR™ (atorvastatin), QUESTRAN™ (cholestyramine), LOLESTID™ (colestipol), TRICOR™ (fenofibrate), LOPID™ (gemfibrate), MEVACOR™ (lovstatin), PRAVACHOL™ (pravastatin), and ZOCOR™ (simvastatin). Non-limiting examples of vasodilating agents include alprostadil, amyl nitrite, PERSANTIN™ (dipyridamole), FLONAN™ (epoprostenol), ISORDIL™ (isosorbide dinitrate), IMDUR™ (isosorbide mononitrate), NIMOTOP™ (nimodipine), INOmax™ (nitric oxide gas), nitroglycerin, papaverine, and PRISCOLINE™ (tolazoline).

In certain embodiments, therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or protease inhibitors (PIs). NRTIs that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, RETROVIR™ (zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC), ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBIVIR™ (zidovudine/lamivudine). NNRTIs that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, VIRAMUNE™ (nevirapine), RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitors that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, CRIXIVAN™ (indinavir), NORVIR™ (ritonavir), INVIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir). In a specific embodiment, antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with Therapeutics of the invention to treat AIDS and/or to prevent or treat HIV infection.

Additional NRTIs include LODENOSINE™ (F-ddA; an acid-stable adenosine NRTI; Triangle/ABBOTT™; COVIRACIL™ (emtricitabine/FTC; structurally related to lamivudine (3TC) but with 3- to 10-fold greater activity in vitro; Triangle/ABBOTT™); dOTC (BCH-10652, also structurally related to lamivudine but retains activity against a substantial proportion of lamivudine-resistant isolates; Biochem Pharma); Adefovir (refused approval for anti-HIV therapy by FDA; Gilead Sciences); PREVEON® (Adefovir Dipivoxil, the active prodrug of adefovir; its active form is PMEA-pp); TENOFOVIR™ (bis-POC PMPA, a PMPA prodrug; Gilead); DAPD/DXG (active metabolite of DAPD; Triangle/ABBOTT™); D-D4FC (related to 3TC, with activity against AZT/3TC-resistant virus); GW420867X (Glaxo Wellcome); ZIAGEN™ (abacavir/159U89; Glaxo Wellcome Inc.); CS-87 (3′ azido-2′,3′-dideoxyuridine; WO 99/66936); and S-acyl-2-thioethyl (SATE)-bearing prodrug forms of β-L-FD4C and β-L-FddC (WO 98/17281).

Additional NNRTIs include COACTINON™ (Emivirine/MKC-442, potent NNRTI of the HEPT class; Triangle/ABBOTT™); CAPRAVIRINE™ (AG-1549/S-1153, a next generation NNRTI with activity against viruses containing the K103N mutation; AGOURON™); PNU-142721 (has 20- to 50-fold greater activity than its predecessor delavirdine and is active against K103N mutants; PHARMACIA™ & Upjohn); DPC-961 and DPC-963 (second-generation derivatives of efavirenz, designed to be active against viruses with the K103N mutation; DUPONT™); GW-420867X (has 25-fold greater activity than HBY097 and is active against K103N mutants; Glaxo Wellcome); CALANOLIDE A (naturally occurring agent from the latex tree; active against viruses containing either or both the Y181C and K103N mutations); and Propolis (WO 99/49830).

Additional protease inhibitors include LOPINAVIR™ (ABT378/r; ABBOTT™ Laboratories); BMS-232632 (an azapeptide; BRISTOL-MYERS SQUIBB™); TIPRANAVIR™ (PNU-140690, a non-peptic dihydropyrone; PHARIVIACIA™ & Upjohn); PD-178390 (a nonpeptidic dihydropyrone; Parke-Davis); BMS 232632 (an azapeptide; BRISTOL-MYERS SQUIBB™); L-756,423 (an indinavir analog; Merck); DMP-450 (a cyclic urea compound; Avid & DUPONT™); AG-1776 (a peptidomimetic with in vitro activity against protease inhibitor-resistant viruses; AGOURON™); VX-175/GW-433908 (phosphate prodrug of amprenavir; Vertex & Glaxo Wellcome); CGP61755 (Ciba); and AGENERASE™ (amprenavir; Glaxo Wellcome Inc.).

Additional antiretroviral agents include fusion inhibitors/gp41 binders. Fusion inhibitors/gp41 binders include T-20 (a peptide from residues 643-678 of the HIV gp41 transmembrane protein ectodomain which binds to gp41 in its resting state and prevents transformation to the fusogenic state; Trimeris) and T-1249 (a second-generation fusion inhibitor; Trimeris).

Additional antiretroviral agents include fusion inhibitors/chemokine receptor antagonists. Fusion inhibitors/chemokine receptor antagonists include CXCR4 antagonists such as AMD 3100 (a bicyclam), SDF-1 and its analogs, and ALX40-4C (a cationic peptide), T22 (an 18 amino acid peptide; Trimeris) and the T22 analogs T134 and T140; CCR5 antagonists such as RANTES (9-68), AOP-RANTES, NNY-RANTES, and TAK-779; and CCR5/CXCR4 antagonists such as NSC 651016 (a distamycin analog). Also included are CCR2B, CCR3, and CCR6 antagonists. Chemokine recpetor agonists such as RANTES, SDF-1, MIP-1α, MIP-1β, etc., may also inhibit fusion.

Additional antiretroviral agents include integrase inhibitors. Integrase inhibitors include dicaffeoylquinic (DFQA) acids; L-chicoric acid (a dicaffeoyltartaric (DCTA) acid); quinalizarin (QLC) and related anthraquinones; ZINTEVIRT™ (AR 177, an oligonucleotide that probably acts at cell surface rather than being a true integrase inhibitor; Arondex); and naphthols such as those disclosed in WO 98/50347.

Additional antiretroviral agents include hydroxyurea-like compounds such as BCX-34 (a purine nucleoside phosphorylase inhibitor; Biocryst); ribonucleotide reductase inhibitors such as DIDOX™ (Molecules for Health); inosine monophosphate dehydrogenase (IMPDH) inhibitors such as VX-497 (Vertex); and mycopholic acids such as CellCept (mycophenolate mofetil; ROCHE™).

Additional antiretroviral agents include inhibitors of viral integrase, inhibitors of viral genome nuclear translocation such as arylene bis(methylketone) compounds; inhibitors of HIV entry such as AOP-RANTES, NNY-RANTES, RANTES-IgG fusion protein, soluble complexes of RANTES and glycosaminoglycans (GAG), and AMD-3100; nucleocapsid zinc finger inhibitors such as dithiane compounds; targets of HIV Tat and Rev; and pharmacoenhancers such as ABT-378.

Other antiretroviral therapies and adjunct therapies include cytokines and lymphokines such as MIP-1α, MIP-1β, SDF-1α, IL-2, PROLEUKIN™ (aldesleukin/L2-7001; CHIRON™), IL-4, IL-10, IL-12, and IL-13; interferons such as IFN-α2a; antagonists of TNFs, NFκB, GM-CSF, M-CSF, and IL-10; agents that modulate immune activation such as cyclosporin and prednisone; vaccines such as Remune™ (HIV Immunogen), APL 400-003 (Apollon), recombinant gp120 and fragments, bivalent (B/E) recombinant envelope glycoprotein, rgp120CM235, MN rgp120, SF-2 rgp120, gp120/soluble CD4 complex, Delta JR-FL protein, branched synthetic peptide derived from discontinuous gp120 C3/C4 domain, fusion-competent immunogens, and Gag, Pol, Nef, and Tat vaccines; gene-based therapies such as genetic suppressor elements (GSEs; WO 98/54366), and intrakines (genetically modified CC chemokines targetted to the ER to block surface expression of newly synthesized CCR5 (Yang et al., PNAS 94:11567-72 (1997); Chen et al., Nat. Med. 3:1110-16 (1997)); antibodies such as the anti-CXCR4 antibody 12G5, the anti-CCR5 antibodies 2D7, 5C7, PA8, PA9, PA10, PA11, PA12, and PA14, the anti-CD4 antibodies Q4120 and RPA-T4, the anti-CCR3 antibody 7B11, the anti-gp120 antibodies 17b, 48d, 447-52D, 257-D, 268-D and 50.1, anti-Tat antibodies, anti-TNF-α antibodies, and monoclonal antibody 33A; aryl hydrocarbon (AH) receptor agonists and antagonists such as TCDD, 3,3′,4,4′,5-pentachlorobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, and α-naphthoflavone (WO 98/30213); and antioxidants such as γ-L-glutamyl-L-cysteine ethyl ester (γ-GCE; WO 99/56764).

In a further embodiment, the Therapeutics of the invention are administered in combination with an antiviral agent. Antiviral agents that may be administered with the Therapeutics of the invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.

In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic agents that may be administered in combination with the Therapeutics of the invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™, ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™, CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™, FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™, PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™ (sargramostim/GM-CSF). In a specific embodiment, Therapeutics of the invention are used in any combination with TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/or ATOVAQUONE™ to prophylactically treat or prevent an opportunistic Pneumocystis carinii pneumonia infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ to prophylactically treat or prevent an opportunistic Mycobacterium avium complex infection. In another specific embodiment, Therapeutics of the invention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ to prophylactically treat or prevent an opportunistic Mycobacterium tuberculosis infection. In another specific embodiment, Therapeutics of the invention are used in any combination with GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat or prevent an opportunistic cytomegalovirus infection. In another specific embodiment, Therapeutics of the invention are used in any combination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ to prophylactically treat or prevent an opportunistic fungal infection. In another specific embodiment, Therapeutics of the invention are used in any combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylactically treat or prevent an opportunistic herpes simplex virus type I and/or type II infection. In another specific embodiment, Therapeutics of the invention are used in any combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ to prophylactically treat or prevent an opportunistic Toxoplasma gondii infection. In another specific embodiment, Therapeutics of the invention are used in any combination with LEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent an opportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention are administered in combination with an antibiotic agent. Antibiotic agents that may be administered with the Therapeutics of the invention include, but are not limited to, amoxicillin, beta-lactamases, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rapamycin, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin.

In other embodiments, the Therapeutics of the invention are administered in combination with immunostimulants. Immunostimulants that may be administered in combination with the Therapeutics of the invention include, but are not limited to, levamisole (e.g., ERGAMISOL™), isoprinosine (e.g. INOSIPLEX™), interferons (e.g. interferon alpha), and interleukins (e.g., IL-2).

In other embodiments, Therapeutics of the invention are administered in combination with immunosuppressive agents. Immunosuppressive agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T cells. Other immunosuppressive agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to, prednisolone, methotrexate, thalidomide, methoxsalen, rapamycin, leflunomide, mizoribine (BREDININ™), brequinar, deoxyspergualin, and azaspirane (SKF 105685), ORTHOCLONE OKT® 3 (muromonab-CD3), SANDIMMUNE™, NEORAL™, SANGDYA™ (cyclosporine), PROGRAF® (FK506, tacrolimus), CELLCEPT® (mycophenolate motefil, of which the active metabolite is mycophenolic acid), IMURAN™ (azathioprine), glucocorticosteroids, adrenocortical steroids such as DELTASONE™ (prednisone) and HYDELTRASOL™ (prednisolone), FOLEX™ and MEXATE™ (methotrxate), OXSORALEN-ULTRA™ (methoxsalen) and RAPAMUNE™ (sirolimus). In a specific embodiment, immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.

In an additional embodiment, Therapeutics of the invention are administered alone or in combination with one or more intravenous immune globulin preparations. Intravenous immune globulin preparations that may be administered with the Therapeutics of the invention include, but not limited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, ATGAM™ (antithymocyte glubulin), and GAMIMUNE™. In a specific embodiment, Therapeutics of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).

In certain embodiments, the Therapeutics of the invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the Therapeutics of the invention include, but are not limited to, corticosteroids (e.g. betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone), nonsteroidal anti-inflammatory drugs (e.g., diclofenac, diflunisal, etodolac, fenoprofen, floctafenine, flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tenoxicam, tiaprofenic acid, and tolmetin.), as well as antihistamines, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and tenidap.

In an additional embodiment, the compositions of the invention are administered alone or in combination with an anti-angiogenic agent. Anti-angiogenic agents that may be administered with the compositions of the invention include, but are not limited to, Angiostatin (ENTREMED™, Rockville, Md.), Troponin-1 (Boston Life Sciences, Boston, Mass.), anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel (TAXOL™), Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, VEGI, Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2, and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.

Representative examples of vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes. Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate. Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes also include oxo complexes. Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes. Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid. Suitable tungsten oxides include tungsten (IV) oxide and tungsten (VI) oxide. Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes. Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates. Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid. Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate. Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilized within the context of the present invention. Representative examples include, but are not limited to, platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al., Cancer Res. 51:22-26, (1991)); Sulphated Polysaccharide Peptidoglycan Complex (SP-PG) (the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate); Staurosporine; modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J. Bio. Chem. 267:17321-17326, (1992)); Chymostatin (Tomkinson et al., Biochem J. 286:475-480, (1992)); Cyclodextrin Tetradecasulfate; Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557, (1990)); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin. Invest. 79:1440-1446, (1987)); anticollagenase-serum; alpha2-antiplasmin (Holmes et al., J. Biol. Chem. 262 (4): 1659-1664, (1987)); Bisantrene (National Cancer Institute); Lob enzarit disodium (N-(2)-carboxyphenyl-4-chloroanthronilic acid disodium or “CCA”; (Takeuchi et al., Agents Actions 36:312-316, (1992)); and metalloproteinase inhibitors such as BB94.

Additional anti-angiogenic factors that may also be utilized within the context of the present invention include Thalidomide, (CELGENE™, Warren, N.J.); Angiostatic steroid; AGM-1470 (H. Brem and J. Folkman J Pediatr. Surg. 28:445-51 (1993)); an integrin alpha v beta 3 antagonist (C. Storgard et al., J. Clin. Invest. 103:47-54 (1999)); carboxynaminolmidazole; Carboxyamidotriazole (CAI) (National Cancer Institute, Bethesda, Md.); Conbretastatin A-4 (CA4P) (OXIGENE™, Boston, Mass.); Squalamine (Magainin Pharmaceuticals, Plymouth Meeting, Pa.); TNP-470, (TAP PHARMACEUTICALS™, Deerfield, Ill.); ZD-0101 ASTRAZENECA™ (London, UK); APRA (CT2584); Benefin, Byrostatin-1 (SC339555); CGP-41251 (PKC 412); CM101; Dexrazoxane (ICRF187); DMXAA; Endostatin; Flavopridiol; Genestein; GTE; ImmTher; Iressa (ZD1839); Octreotide (Somatostatin); Panretin; Penacillamine; Photopoint; PI-88; Prinomastat (AG-3340) Purlytin; Suradista (FCE26644); Tamoxifen (NOLVADEX™); Tazarotene; Tetrathiomolybdate; XELODA™ (Capecitabine); and 5-Fluorouracil.

Anti-angiogenic agents that may be administed in combination with the compounds of the invention may work through a variety of mechanisms including, but not limited to, inhibiting proteolysis of the extracellular matrix, blocking the function of endothelial cell-extracellular matrix adhesion molecules, by antagonizing the function of angiogenesis inducers such as growth factors, and inhibiting integrin receptors expressed on proliferating endothelial cells. Examples of anti-angiogenic inhibitors that interfere with extracellular matrix proteolysis and which may be administered in combination with the compositons of the invention include, but are not lmited to, AG-3340 (AGOURON™, La Jolla, Calif.), BAY-12-9566 (BAYER™, West Haven, Conn.), BMS-275291 (BRISTOL-MYERS SQUIBB™, Princeton, N.J.), CGS-27032A (NOVARTIS™, East Hanover, N.J.), Marimastat (British Biotech, Oxford, UK), and METASTAT™ (AETERNA™, St-Foy, Quebec). Examples of anti-angiogenic inhibitors that act by blocking the function of endothelial cell-extracellular matrix adhesion molecules and which may be administered in combination with the compositons of the invention include, but are not lmited to, EMD-121974 (MERCK™ KcgaA Darmstadt, Germany) and VITAXIN™ (IXSYS™, La Jolla, Calif./MEDIMMUNE™, Gaithersburg, Md.). Examples of anti-angiogenic agents that act by directly antagonizing or inhibiting angiogenesis inducers and which may be administered in combination with the compositons of the invention include, but are not lmited to, Angiozyme (Ribozyme, Boulder, Colo.), Anti-VEGF antibody (GENENTECH™, S. San Francisco, Calif.), PTK-787/ZK-225846 (NOVARTIS™, Basel, Switzerland), SU-101 (SUGEN™, S. San Francisco, Calif.), SU-5416 (SUGEN™/PHARMACIA™ Upjohn, Bridgewater, N.J.), and SU-6668 (SUGEN™). Other anti-angiogenic agents act to indirectly inhibit angiogenesis. Examples of indirect inhibitors of angiogenesis which may be administered in combination with the compositons of the invention include, but are not limited to, IM-862 (CYTRAN™, Kirkland, Wash.), Interferon-alpha, IL-12 (ROCHE™, Nutley, N.J.), and Pentosan polysulfate (Georgetown University, Washington, D.C.).

In particular embodiments, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of an autoimmune disease, such as for example, an autoimmune disease described herein.

In a particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of arthritis. In a more particular embodiment, the use of compositions of the invention in combination with anti-angiogenic agents is contemplated for the treatment, prevention, and/or amelioration of rheumatoid arthritis.

In another embodiment, the polynucleotides encoding a polypeptide of the present invention are administered in combination with an angiogenic protein, or polynucleotides encoding an angiogenic protein. Examples of angiogenic proteins that may be administered with the compositions of the invention include, but are not limited to, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2, VEGF-3, epidermal growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor, tumor necrosis factor alpha, hepatocyte growth factor, insulin-like growth factor, colony stimulating factor, macrophage colony stimulating factor, granulocyte/macrophage colony stimulating factor, and nitric oxide synthase.

In additional embodiments, compositions of the invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the Therapeutics of the invention include, but are not limited to alkylating agents such as nitrogen mustards (for example, Mechlorethamine, cyclophosphamide, Cyclophosphamide Ifosfamide, Melphalan (L-sarcolysin), and Chlorambucil), ethylenimines and methylmelamines (for example, Hexamethylmelamine and Thiotepa), alkyl sulfonates (for example, Busulfan), nitrosoureas (for example, Carmustine (BCNU), Lomustine (CCNU), Semustine (methyl-CCNU), and Streptozocin (streptozotocin)), triazenes (for example, Dacarbazine (DTIC; dimethyltriazenoimidazolecarboxamide)), folic acid analogs (for example, Methotrexate (amethopterin)), pyrimidine analogs (for example, Fluorouacil (5-fluorouracil; 5-FU), Floxuridine (fluorodeoxyuridine; FudR), and Cytarabine (cytosine arabinoside)), purine analogs and related inhibitors (for example, Mercaptopurine (6-mercaptopurine; 6-MP), Thioguanine (6-thioguanine; TG), and Pentostatin (2′-deoxycoformycin)), vinca alkaloids (for example, Vinblastine (VLB, vinblastine sulfate)) and Vincristine (vincristine sulfate)), epipodophyllotoxins (for example, Etoposide and Teniposide), antibiotics (for example, Dactinomycin (actinomycin D), Daunorubicin (daunomycin; rubidomycin), Doxorubicin, Bleomycin, Plicamycin (mithramycin), and Mitomycin (mitomycin C), enzymes (for example, L-Asparaginase), biological response modifiers (for example, Interferon-alpha and interferon-alpha-2b), platinum coordination compounds (for example, Cisplatin (cis-DDP) and Carboplatin), anthracenedione (Mitoxantrone), substituted ureas (for example, Hydroxyurea), methylhydrazine derivatives (for example, Procarbazine (N-methylhydrazine; MIH), adrenocorticosteroids (for example, Prednisone), progestins (for example, Hydroxyprogesterone caproate, Medroxyprogesterone, Medroxyprogesterone acetate, and Megestrol acetate), estrogens (for example, Diethylstilbestrol (DES), Diethylstilbestrol diphosphate, Estradiol, and Ethinyl estradiol), antiestrogens (for example, Tamoxifen), androgens (Testosterone proprionate, and Fluoxymesterone), antiandrogens (for example, Flutamide), gonadotropin-releasing horomone analogs (for example, Leuprolide), other hormones and hormone analogs (for example, methyltestosterone, estramustine, estramustine phosphate sodium, chlorotrianisene, and testolactone), and others (for example, dicarbazine, glutamic acid, and mitotane).

In one embodiment, the compositions of the invention are administered in combination with one or more of the following drugs: infliximab (also known as Remicade™ Centocor, Inc.), Trocade (ROCHE™, RO-32-3555), Leflunomide (also known as Arava™ from HOECHST MARION ROUSSEL™), Kineret™ (an IL-1 Receptor antagonist also known as Anakinra from AMGEN™, Inc.)

In a specific embodiment, compositions of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or combination of one or more of the components of CHOP. In one embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies, human monoclonal anti-CD20 antibodies. In another embodiment, the compositions of the invention are administered in combination with anti-CD20 antibodies and CHOP, or anti-CD20 antibodies and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with Rituximab. In a further embodiment, compositions of the invention are administered with Rituximab and CHOP, or Rituximab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. In a specific embodiment, compositions of the invention are administered in combination with tositumomab. In a further embodiment, compositions of the invention are administered with tositumomab and CHOP, or tositumomab and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. The anti-CD20 antibodies may optionally be associated with radioisotopes, toxins or cytotoxic prodrugs.

In another specific embodiment, the compositions of the invention are administered in combination ZEVALIN™. In a further embodiment, compositions of the invention are administered with ZEVALIN™ and CHOP, or ZEVALIN™ and any combination of one or more of the components of CHOP, particularly cyclophosphamide and/or prednisone. ZEVALIN™ may be associated with one or more radisotopes. Particularly preferred isotopes are ⁹⁰Y and ¹¹¹In.

In an additional embodiment, the Therapeutics of the invention are administered in combination with cytokines Cytokines that may be administered with the Therapeutics of the invention include, but are not limited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment, Therapeutics of the invention may be administered with any interleukin, including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In one embodiment, the Therapeutics of the invention are administered in combination with members of the TNF family. TNF, TNF-related or TNF-like molecules that may be administered with the Therapeutics of the invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), endokine-alpha (International Publication No. WO 98/07880), TR6 (International Publication No. WO 98/30694), OPG, and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095), DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), and TR12, and soluble forms CD154, CD70, and CD153.

In an additional embodiment, the Therapeutics of the invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the Therapeutics of the invention include, but are not limited to, Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PlGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (PlGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96/39515; Vascular Endothelial Growth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186 (VEGF-B186), as disclosed in International Publication Number WO 96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/02543; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed in International Publication Number WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E), as disclosed in German Patent Number DE19639601. The above mentioned references are herein incorporated by reference in their entireties.

In an additional embodiment, the Therapeutics of the invention are administered in combination with Fibroblast Growth Factors. Fibroblast Growth Factors that may be administered with the Therapeutics of the invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, and FGF-15.

In an additional embodiment, the Therapeutics of the invention are administered in combination with hematopoietic growth factors. Hematopoietic growth factors that may be administered with the Therapeutics of the invention include, but are not limited to, granulocyte macrophage colony stimulating factor (GM-CSF) (sargramostim, LEUKINE™, PROKINE™), granulocyte colony stimulating factor (G-CSF) (filgrastim, NEUPOGEN™), macrophage colony stimulating factor (M-CSF, CSF-1) erythropoietin (epoetin alfa, EPOGEN™, PROCRIT™), stem cell factor (SCF, c-kit ligand, steel factor), megakaryocyte colony stimulating factor, PIXY321 (a GMCSF/IL-3 fusion protein), interleukins, especially any one or more of IL-1 through IL-12, interferon-gamma, or thrombopoietin.

In certain embodiments, Therapeutics of the present invention are administered in combination with adrenergic blockers, such as, for example, acebutolol, atenolol, betaxolol, bisoprolol, carteolol, osmolol, labetalol, metoprolol, nadolol, oxprenolol, penbutolol, pindolol, propranolol, sotalol, and timolol.

In another embodiment, the Therapeutics of the invention are administered in combination with an antiarrhythmic drug (e.g., adenosine, amidoarone, bretylium, digitalis, digoxin, digitoxin, diliazem, disopyramide, esmolol, flecamide, lidocaine, mexiletine, moricizine, phenyloin, procainamide, N-acetyl procainamide, propafenone, propranolol, quinidine, sotalol, tocamide, and verapamil).

In another embodiment, the Therapeutics of the invention are administered in combination with diuretic agents, such as carbonic anhydrase-inhibiting agents (e.g., acetazolamide, dichlorphenamide, and methazolamide), osmotic diuretics (e.g., glycerin, isosorbide, mannitol, and urea), diuretics that inhibit Na⁺—K-2Cl⁻ symport (e.g., furosemide, bumetanide, azosemide, piretanide, tripamide, ethacrynic acid, muzolimine, and torsemide), thiazide and thiazide-like diuretics (e.g., bendroflumethiazide, benzthiazide, chlorothiazide, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, polythiazide, trichormethiazide, chlorthalidone, indapamide, metolazone, and quinethazone), potassium sparing diuretics (e.g., amiloride and triamterene), and mineralcorticoid receptor antagonists (e.g., spironolactone, canrenone, and potassium canrenoate).

In one embodiment, the Therapeutics of the invention are administered in combination with treatments for endocrine and/or hormone imbalance disorders. Treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, ¹²⁷I, radioactive isotopes of iodine such as ¹³¹I and ¹²³I; recombinant growth hormone, such as HUMATROPE™ (recombinant somatropin); growth hormone analogs such as PROTROPIN™ (somatrem); dopamine agonists such as PARLODEL™ (bromocriptine); somatostatin analogs such as SANDOSTATIN™ (octreotide); gonadotropin preparations such as PREGNYL™, A.P.L.™ and PROFASI™ (chorionic gonadotropin (CG)), PERGONAL™ (menotropins), and METRODIN™ (urofollitropin (uFSH)); synthetic human gonadotropin releasing hormone preparations such as FACTREL™ and LUTREPULSE™ (gonadorelin hydrochloride); synthetic gonadotropin agonists such as LUPRON™ (leuprolide acetate), SUPPRELIN™ (histrelin acetate), SYNAREL™ (nafarelin acetate), and ZOLADEX™ (goserelin acetate); synthetic preparations of thyrotropin-releasing hormone such as RELEFACT TRH™ and THYPINONE™ (protirelin); recombinant human TSH such as THYROGEN™; synthetic preparations of the sodium salts of the natural isomers of thyroid hormones such as L-T₄™, SYNTHROID™ and LEVOTHROID™ (levothyroxine sodium), L-T₃™, CYTOMEL™ and TRIOSTAT™ (liothyroine sodium), and THYROLAR™ (liotrix); antithyroid compounds such as 6-n-propylthiouracil (propylthiouracil), 1-methyl-2-mercaptoimidazole and TAPAZOLE™ (methimazole), NEO-MERCAZOLE™ (carbimazole); beta-adrenergic receptor antagonists such as propranolol and esmolol; Ca²⁺ channel blockers; dexamethasone and iodinated radiological contrast agents such as TELEPAQUE™ (iopanoic acid) and ORAGRAFIN™ (sodium ipodate).

Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, estrogens or congugated estrogens such as ESTRACE™ (estradiol), ESTINYL™ (ethinyl estradiol), PREMARIN™, ESTRATAB™, ORTHO-EST™, OGEN™ and estropipate (estrone), ESTROVIS™ (quinestrol), ESTRADERM™ (estradiol), DELESTROGEN™ and VALERGEN™ (estradiol valerate), DEPO-ESTRADIOL CYPIONATE™ and ESTROJECT LA™ (estradiol cypionate); antiestrogens such as NOLVADEX™ (tamoxifen), SEROPHENE™ and CLOMID™ (clomiphene); progestins such as DURALUTIN™ (hydroxyprogesterone caproate), MPA™ and DEPO—PROVERA™ (medroxyprogesterone acetate), PROVERA™ and CYCRIN™ (MPA), MEGACE™ (megestrol acetate), NORLUTIN™ (norethindrone), and NORLUTATE™ and AYGESTIN™ (norethindrone acetate); progesterone implants such as NORPLANT SYSTEM™ (subdermal implants of norgestrel); antiprogestins such as RU 486™ (mifepristone); hormonal contraceptives such as ENOVID™ (norethynodrel plus mestranol), PROGESTASERT™ (intrauterine device that releases progesterone), LOESTRIN™, BREVICON™, MODICON™, GENORA™, NELONA™, NORINYL™, OVACON-35™ and OVACON-50™ (ethinyl estradiol/norethindrone), LEVLEN™, NORDETTE™, TRI-LEVLEN™ and TRIPHASIL-21™ (ethinyl estradiol/levonorgestrel) LO/OVRAL™ and OVRAL™ (ethinyl estradiol/norgestrel), DEMULEN™ (ethinyl estradiol/ethynodiol diacetate), NORINYL™, ORTHO-NOVUM™, NORETHIN™, GENORA™, and NELOVA™ (norethindrone/mestranol), DESOGEN™ and ORTHO-CEPT™ (ethinyl estradiol/desogestrel), ORTHO-CYCLEN™ and ORTHO-TRICYCLEN™ (ethinyl estradiol/norgestimate), MICRONOR™ and NOR-QD™ (norethindrone), and OVRETTE™ (norgestrel).

Additional treatments for endocrine and/or hormone imbalance disorders include, but are not limited to, testosterone esters such as methenolone acetate and testosterone undecanoate; parenteral and oral androgens such as TESTOJECT-50™ (testosterone), TESTEX™ (testosterone propionate), DELATESTRYL™ (testosterone enanthate), DEPO-TESTOSTERONE™ (testosterone cypionate), DANOCRINE™ (danazol), HALOTESTIN™ (fluoxymesterone), ORETON METHYL™, TESTRED™ and VIRILON™ (methyltestosterone), and OXANDRIN™ (oxandrolone); testosterone transdermal systems such as TESTODERM™; androgen receptor antagonist and 5-alpha-reductase inhibitors such as ANDROCUR™ (cyproterone acetate), EULEXIN™ (flutamide), and PROSCAR™ (finasteride); adrenocorticotropic hormone preparations such as CORTROSYN™ (cosyntropin); adrenocortical steroids and their synthetic analogs such as ACLOVATE™ (alclometasone dipropionate), CYCLOCORT™ (amcinonide), BECLOVENT™ and VANCERIL™ (beclomethasone dipropionate), CELESTONE™ (betamethasone), BENISONE™ and UTICORT™ (betamethasone benzoate), DIPROSONE™ (betamethasone dipropionate), CELESTONE PHOSPHATE™ (betamethasone sodium phosphate), CELESTONE SOLUSPAN™ (betamethasone sodium phosphate and acetate), BETA-VALT™ and VALISONE™ (betamethasone valerate), TEMOVATE™ (clobetasol propionate), CLODERM™ (clocortolone pivalate), CORTEF™ and HYDROCORTONE™ (cortisol (hydrocortisone)), HYDROCORTONE ACETATE™ (cortisol (hydrocortisone) acetate), LOCOID™ (cortisol (hydrocortisone) butyrate), HYDROCORTONE PHOSPHATE™ (cortisol (hydrocortisone) sodium phosphate), A-HYDROCORT™ and SOLU CORTEF™ (cortisol (hydrocortisone) sodium succinate), WESTCORT™ (cortisol (hydrocortisone) valerate), CORTISONE ACETATE™ (cortisone acetate), DESOWEN™ and TRIDESILON™ (desonide), TOPICORT™ (desoximetasone), DECADRON™ (dexamethasone), DECADRON LA™ (dexamethasone acetate), DECADRON PHOSPHATE™ and HEXADROL PHOSPHATE™ (dexamethasone sodium phosphate), FLORONE™ and MAXIFLOR™ (diflorasone diacetate), FLORINEF ACETATE™ (fludrocortisone acetate), AEROBID™ and NASALIDE™ (flunisolide), FLUONID™ and SYNALAR™ (fluocinolone acetonide), LIDEX™ (fluocinonide), FLUOR-OP™ and FML™ (fluorometholone), CORDRAN™ (flurandrenolide), HALOG™ (halcinonide), HMS LIZUIFILM™ (medrysone), MEDROL™ (methylprednisolone), DEPO-MEDROL™ and MEDROL ACETATE™ (methylprednisone acetate), A-METHAPRED™ and SOLUMEDROL™ (methylprednisolone sodium succinate), ELOCON™ (mometasone furoate), HALDRONE™ (paramethasone acetate), DELTA-CORTEF™ (prednisolone), ECONOPRED™ (prednisolone acetate), HYDELTRASOL™ (prednisolone sodium phosphate), HYDELTRA-T.B.A™ (prednisolone tebutate), DELTASONE™ (prednisone), ARISTOCORT™ and KENACORT™ (triamcinolone), KENALOG™ (triamcinolone acetonide), ARISTOCORT™ and KENACORT DIACETATE™ (triamcinolone diacetate), and ARISTOSPAN™ (triamcinolone hexacetonide); inhibitors of biosynthesis and action of adrenocortical steroids such as CYTADREN™ (aminoglutethimide), NIZORAL™ (ketoconazole), MODRASTANE™ (trilostane), and METOPIRONE™ (metyrapone); bovine, porcine or human insulin or mixtures thereof; insulin analogs; recombinant human insulin such as HUMULIN™ and NOVOLIN™; oral hypoglycemic agents such as ORAMIDE™ and ORINASE™ (tolbutamide), DIABINESE™ (chlorpropamide), TOLAMIDE™ and TOLINASE™ (tolazamide), DYMELOR™ (acetohexamide), glibenclamide, MICRONASE™, DIBETA™ and GLYNASE™ (glyburide), GLUCOTROL™ (glipizide), and DIAMICRON™ (gliclazide), GLUCOPHAGE™ (metformin), PRECOSE™ (acarbose), AMARYL™ (glimepiride), and ciglitazone; thiazolidinediones (TZDs) such as rosiglitazone, AVANDIA™ (rosiglitazone maleate), ACTOS™ (piogliatazone), troglitazone, ciglitazone, pioglitazone, and alpha-glucosidase inhibitors; bovine or porcine glucagon; somatostatins such as SANDOSTATIN™ (octreotide); and diazoxides such as PROGLYCEM™ (diazoxide). In still other embodiments, Therapeutics of the invention are administered in combination with one or more of the following: a biguanide antidiabetic agent, a glitazone antidiabetic agent, and a sulfonylurea antidiabetic agent.

In one embodiment, the Therapeutics of the invention are administered in combination with treatments for uterine motility disorders. Treatments for uterine motility disorders include, but are not limited to, estrogen drugs such as conjugated estrogens (e.g., PREMARIN® and ESTRATAB®), estradiols (e.g., CLIMARA® and ALORA®), estropipate, and chlorotrianisene; progestin drugs (e.g., AMEN® (medroxyprogesterone), MICRONOR® (norethidrone acetate), PROMETRIUM® progesterone, and megestrol acetate); and estrogen/progesterone combination therapies such as, for example, conjugated estrogens/medroxyprogesterone (e.g., PREMPRO™ and PREMPHASE) and norethindrone acetate/ethinyl estsradiol (e.g., FEMHRT™).

In an additional embodiment, the Therapeutics of the invention are administered in combination with drugs effective in treating iron deficiency and hypochromic anemias, including but not limited to, ferrous sulfate (iron sulfate, FEOSOL™), ferrous fumarate (e.g., FEOSTAT™), ferrous gluconate (e.g., FERGON™), polysaccharide-iron complex (e.g., NIFEREX™), iron dextran injection (e.g., INFED™), cupric sulfate, pyroxidine, riboflavin, Vitamin B₁₂, cyancobalamin injection (e.g., REDISOL™, RUBRAMIN PC™), hydroxocobalamin, folic acid (e.g., FOLVITE™), leucovorin (folinic acid, 5-CHOH4PteGlu, citrovorum factor) or WELLCOVORIN (Calcium salt of leucovorin), transferrin or ferritin.

In certain embodiments, the Therapeutics of the invention are administered in combination with agents used to treat psychiatric disorders. Psychiatric drugs that may be administered with the Therapeutics of the invention include, but are not limited to, antipsychotic agents (e.g., chlorpromazine, chlorprothixene, clozapine, fluphenazine, haloperidol, loxapine, mesoridazine, molindone, olanzapine, perphenazine, pimozide, quetiapine, risperidone, thioridazine, thiothixene, trifluoperazine, and triflupromazine), antimanic agents (e.g., carbamazepine, divalproex sodium, lithium carbonate, and lithium citrate), antidepressants (e.g., amitriptyline, amoxapine, bupropion, citalopram, clomipramine, desipramine, doxepin, fluvoxamine, fluoxetine, imipramine, isocarboxazid, maprotiline, mirtazapine, nefazodone, nortriptyline, paroxetine, phenelzine, protriptyline, sertraline, tranylcypromine, trazodone, trimipramine, and venlafaxine), antianxiety agents (e.g., alprazolam, buspirone, chlordiazepoxide, clorazepate, diazepam, halazepam, lorazepam, oxazepam, and prazepam), and stimulants (e.g., d-amphetamine, methylphenidate, and pemoline).

In other embodiments, the Therapeutics of the invention are administered in combination with agents used to treat neurological disorders. Neurological agents that may be administered with the Therapeutics of the invention include, but are not limited to, antiepileptic agents (e.g., carbamazepine, clonazepam, ethosuximide, phenobarbital, phenyloin, primidone, valproic acid, divalproex sodium, felbamate, gabapentin, lamotrigine, levetiracetam, oxcarbazepine, tiagabine, topiramate, zonisamide, diazepam, lorazepam, and clonazepam), antiparkinsonian agents (e.g., levodopa/carbidopa, selegiline, amantidine, bromocriptine, pergolide, ropinirole, pramipexole, benztropine; biperiden; ethopropazine; procyclidine; trihexyphenidyl, tolcapone), and ALS therapeutics (e.g. riluzole).

In another embodiment, Therapeutics of the invention are administered in combination with vasodilating agents and/or calcium channel blocking agents. Vasodilating agents that may be administered with the Therapeutics of the invention include, but are not limited to, Angiotensin Converting Enzyme (ACE) inhibitors (e.g., papaverine, isoxsuprine, benazepril, captopril, cilazapril, enalapril, enalaprilat, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, spirapril, trandolapril, and nylidrin), and nitrates (e.g., amyl nitrite, isosorbide dinitrate, isosorbide mononitrate, nitric oxide gas, and nitroglycerin). Examples of calcium channel blocking agents that may be administered in combination with the Therapeutics of the invention include, but are not limited to amlodipine, bepridil, diltiazem, felodipine, flunarizine, isradipine, nicardipine, nifedipine, nimodipine, and verapamil.

Other vasodilating agents that may be administered with the Therapeutics of the invention include, but are not limited to, epoprostenol and alprostadil.

[1091] In certain embodiments, the Therapeutics of the invention are administered in combination with treatments for cardiovascular disorders. Treatments for cardiovascular disorders that may be administered with the Therapeutic of the invention include, but are not limited to, angiotensin II blockers (e.g., irbesartan, losartan, and valsartan), alpha adrenergic blockers (e.g., doxazosin, prazosin, tamsulosin, and terazosin), hypotensive agents (e.g., clonidine, hydralazine, methyldopa, minoxidil, nitroprusside, and reserpine) and antilipemic agents (e.g., atorvastatin, cholestryamine, colestipol, fenofibrate, gemfibrate, lovstatin, pravastatin, and simvastatin).

In certain embodiments, the Therapeutics of the invention are administered in combination with treatments for gastrointestinal disorders. Treatments for gastrointestinal disorders that may be administered with the Therapeutic of the invention include, but are not limited to, H₂ histamine receptor antagonists (e.g., TAGAMET™ (cimetidine), ZANTAC™ (ranitidine), PEPCID™ (famotidine), and AXID™ (nizatidine)); inhibitors of H⁺, K⁺ ATPase (e.g., PREVACID™ (lansoprazole) and PRILOSEC™ (omeprazole)); Bismuth compounds (e.g., PEPTO-BISMOL™ (bismuth subsalicylate) and DE-NOL™ (bismuth subcitrate)); various antacids; sucralfate; prostaglandin analogs (e.g. CYTOTEC™ (misoprostol)); muscarinic cholinergic antagonists; laxatives (e.g., surfactant laxatives, stimulant laxatives, saline and osmotic laxatives); antidiarrheal agents (e.g., LOMOTIL™ (diphenoxylate), MOTOFEN™ (diphenoxin), and IMODIUM™ (loperamide hydrochloride)), synthetic analogs of somatostatin such as SANDOSTATIN™ (octreotide), antiemetic agents (e.g., ZOFRAN™ (ondansetron), KYTRIL™ (granisetron hydrochloride), tropisetron, dolasetron, metoclopramide, chlorpromazine, perphenazine, prochlorperazine, promethazine, thiethylperazine, triflupromazine, domperidone, haloperidol, droperidol, trimethobenzamide, dexamethasone, methylprednisolone, dronabinol, and nabilone); D2 antagonists (e.g., metoclopramide, trimethobenzamide and chlorpromazine); bile salts; chenodeoxycholic acid; ursodeoxycholic acid; and pancreatic enzyme preparations such as pancreatin and pancrelipase.

In additional embodiments, the Therapeutics of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

Example 14 Method of Treating Decreased Levels of the Polypeptide

The present invention relates to a method for treating an individual in need of an increased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of polypeptides (including agonists thereto), and/or antibodies of the invention. Moreover, it will be appreciated that conditions caused by a decrease in the standard or normal expression level of a polypeptide of the present invention in an individual may be treated by administering agonists of said polypeptide. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a Therapeutic comprising an amount of the agonist (including polypeptides and antibodies of the present invention) to increase the activity level of the polypeptide in such an individual.

For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 μg/kg of the agonist for six consecutive days. The exact details of the dosing scheme, based on administration and formulation, are provided in Example 13.

Example 15 Method of Treating Increased Levels of the Polypeptide

The present invention also relates to a method of treating an individual in need of a decreased level of a polypeptide of the invention in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of the invention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, due to a variety of etiologies, such as cancer.

For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The antisense polynucleotides of the present invention can be formulated using techniques and formulations described herein (e.g. see Example 13), or otherwise known in the art.

Example 16 Method of Treatment Using Gene Therapy-Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37 degree C. for approximately one week.

At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads.

The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively as set forth in Example 1 using primers and having appropriate restriction sites and initiation/stop codons, if necessary. Preferably, the 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced.

The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.

Example 17 Gene Therapy Using Endogenous Genes Corresponding to Polynucleotides of the Invention

Another method of gene therapy according to the present invention involves operably associating the endogenous polynucleotide sequence of the invention with a promoter via homologous recombination as described, for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication NO: WO 96/29411, published Sep. 26, 1996; International Publication NO: WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); and Zijlstra et al., Nature, 342:435-438 (1989). This method involves the activation of a gene which is present in the target cells, but which is not expressed in the cells, or is expressed at a lower level than desired.

Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5′ non-coding sequence of endogenous polynucleotide sequence, flanking the promoter. The targeting sequence will be sufficiently near the 5′ end of the polynucleotide sequence so the promoter will be operably linked to the endogenous sequence upon homologous recombination. The promoter and the targeting sequences can be amplified using PCR. Preferably, the amplified promoter contains distinct restriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ end of the first targeting sequence contains the same restriction enzyme site as the 5′ end of the amplified promoter and the 5′ end of the second targeting sequence contains the same restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase. The digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The construct is size fractionated on an agarose gel, then purified by phenol extraction and ethanol precipitation.

In this Example, the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynucleotide constructs may also be administered with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, precipitating agents, etc. Such methods of delivery are known in the art.

Once the cells are transfected, homologous recombination will take place which results in the promoter being operably linked to the endogenous polynucleotide sequence. This results in the expression of polynucleotide corresponding to the polynucleotide in the cell. Expression may be detected by immunological staining, or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM+10% fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot of the cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin. The final cell suspension contains approximately 3×10⁶ cells/ml. Electroporation should be performed immediately following resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus corresponding to the polynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site on the 5′ end and a BamHI site on the 3′ end. Two non-coding sequences are amplified via PCR: one non-coding sequence (fragment 1) is amplified with a HindIII site at the 5′ end and an Xba site at the 3′ end; the other non-coding sequence (fragment 2) is amplified with a BamHI site at the 5′ end and a HindIII site at the 3′ end. The CMV promoter and the fragments (1 and 2) are digested with the appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI; fragment 2-BamHI) and ligated together. The resulting ligation product is digested with HindIII, and ligated with the HindIII-digested pUC18 plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 μg/ml. 0.5 ml of the cell suspension (containing approximately 1.5.×10⁶ cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed. Electroporation is performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and 250-300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature for approximately 5 min, and the contents of the cuvette are then gently removed with a sterile transfer pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at 37 degree C. The following day, the media is aspirated and replaced with 10 ml of fresh media and incubated for a further 16-24 hours.

The engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. The fibroblasts now produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 18 Method of Treatment Using Gene Therapy—in Vivo

Another aspect of the present invention is using in vivo gene therapy methods to prevent, treat, and/or ameliorate diseases, disorders, and conditions (such as immune, cardiovascular, cancer, and other proliferative diseases, disorders, and conditions). The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. The polynucleotide of the present invention may be operatively linked to (i.e., associated with) a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. No. 5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res. 35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997); Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., Gene Ther. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290 (1996) (incorporated herein by reference).

The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, LIPOFECTIN™ or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods well known to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months.

The polynucleotide construct can be delivered to the interstitial space of tissues within an animal, including muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides.

For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 g/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin™. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 μm cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experimentation in mice can be used to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA.

Example 19 Transgenic Animals

The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.

Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety.

Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.

Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.

Example 20 Knock-Out Animals

Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (e.g., see Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.

In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form that, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.

Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.

Example 21 Assays Detecting Stimulation or Inhibition of B Cell Proliferation and Differentiation

Generation of functional humoral immune responses requires both soluble and cognate signaling between B-lineage cells and their microenvironment. Signals may impart a positive stimulus that allows a B-lineage cell to continue its programmed development, or a negative stimulus that instructs the cell to arrest its current developmental pathway. To date, numerous stimulatory and inhibitory signals have been found to influence B cell responsiveness including IL-2, IL-4, IL-5, IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signals are by themselves weak effectors but can, in combination with various co-stimulatory proteins, induce activation, proliferation, differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is the TNF-superfamily. Within this family CD40, CD27, and CD30 along with their respective ligands CD154, CD70, and CD153 have been found to regulate a variety of immune responses. Assays which allow for the detection and/or observation of the proliferation and differentiation of these B-cell populations and their precursors are valuable tools in determining the effects various proteins may have on these B-cell populations in terms of proliferation and differentiation. Listed below are two assays designed to allow for the detection of the differentiation, proliferation, or inhibition of B-cell populations and their precursors.

In Vitro Assay—Agonists or antagonists of the invention can be assessed for its ability to induce activation, proliferation, differentiation or inhibition and/or death in B-cell populations and their precursors. The activity of the agonists or antagonists of the invention on purified human tonsillar B cells, measured qualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessed in a standard B-lymphocyte co-stimulation assay in which purified tonsillar B cells are cultured in the presence of either formalin-fixed Staphylococcus aureus Cowan I (SAC) or immobilized anti-human IgM antibody as the priming agent. Second signals such as IL-2 and IL-15 synergize with SAC and IgM crosslinking to elicit B cell proliferation as measured by tritiated-thymidine incorporation. Novel synergizing agents can be readily identified using this assay. The assay involves isolating human tonsillar B cells by magnetic bead (MACS) depletion of CD3-positive cells. The resulting cell population is greater than 95% B cells as assessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a 96-well plate to which are added 10⁵ B-cells suspended in culture medium (RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin, 10 μg/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of 150 μA. Proliferation or inhibition is quantitated by a 20 h pulse (1 uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition. The positive and negative controls are IL2 and medium respectively.

In vivo Assay—BALB/c mice are injected (i.p.) twice per day with buffer only, or 2 mg/Kg of agonists or antagonists of the invention, or truncated forms thereof. Mice receive this treatment for 4 consecutive days, at which time they are sacrificed and various tissues and serum collected for analyses. Comparison of H&E sections from normal spleens and spleens treated with agonists or antagonists of the invention identify the results of the activity of the agonists or antagonists on spleen cells, such as the diffusion of peri-arterial lymphatic sheaths, and/or significant increases in the nucleated cellularity of the red pulp regions, which may indicate the activation of the differentiation and proliferation of B-cell populations. Immunohistochemical studies using a B cell marker, anti-CD45R(B220), are used to determine whether any physiological changes to splenic cells, such as splenic disorganization, are due to increased B-cell representation within loosely defined B-cell zones that infiltrate established T-cell regions.

Flow cytometric analyses of the spleens from mice treated with agonist or antagonist are used to indicate whether the agonists or antagonists specifically increases the proportion of ThB+, CD45R(B220)dull B cells over that which is observed in control mice.

Likewise, a predicted consequence of increased mature B-cell representation in vivo is a relative increase in serum Ig titers. Accordingly, serum IgM and IgA levels are compared between buffer and agonists or antagonists-treated mice.

The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).

Example 22 T Cell Proliferation Assay

Proliferation Assay for Resting PBLs

A CD3-induced proliferation assay is performed on PBMCs and is measured by the uptake of ³H-thymidine. The assay is performed as follows. Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a, Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4° C. (1 μg/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three times with PBS. PBMC are isolated by F/H gradient centrifugation from human peripheral blood and added to quadruplicate wells (5×10⁴/well) of mAb coated plates in RPMI containing 10% FCS and P/S in the presence of varying concentrations of agonists or antagonists of the invention (total volume 200 μl). Relevant protein buffer and medium alone are controls. After 48 hr. culture at 37° C., plates are spun for 2 min. at 1000 rpm and 100 μl of supernatant is removed and stored at −20° C. for measurement of IL-2 (or other cytokines) if effect on proliferation is observed. Wells are supplemented with 100 μl of medium containing 0.5 μCi of ³H-thymidine and cultured at 37° C. for 18-24 hr. Wells are harvested and incorporation of ³H-thymidine used as a measure of proliferation. Anti-CD3 alone is the positive control for proliferation. IL-2 (100 U/ml) is also used as a control which enhances proliferation. Control antibody which does not induce proliferation of T cells is used as the negative control for the effects of agonists or antagonists of the invention.

Alternatively, a proliferation assay on resting PBL (peripheral blood lymphocytes) is measured by the up-take of ³H-thymidine. The assay is performed as follows. PBMC are isolated by FICOLL™ (LSM, ICN Biotechnologies, Aurora, Ohio) gradient centrifugation from human peripheral blood, and are cultured overnight in 10% (Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight incubation period allows the adherent cells to attach to the plastic, which results in a lower background in the assay as there are fewer cells that can act as antigen presenting cells or that might be producing growth factors. The following day the non-adherent cells are collected, washed and used in the proliferation assay. The assay is performed in a 96 well plate using 2×10⁴ cells/well in a final volume of 200 μl. The supernatants (e.g., CHO or 293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 60 μl are added to 140 μl of 10% FCS/RPMI containing the cells. Control supernatants are used at the same final dilution and express the following proteins: vector (negative control), IL-2 (*), IFNγ, TNFα, IL-10 and TR2. In addition to the control supernatants, recombinant human IL-2 (R & D Systems, Minneapolois, Minn.) at a final concentration of 100 ng/ml is also used. After 24 hours of culture, each well is pulsed with 1 μCi of ³H-thymidine (Nen, Boston, Mass.). Cells are then harvested 20 hours following pulsing and incorporation of ³H-thymidine is used as a measure of proliferation. Results are expressed as an average of triplicate samples plus or minus standard error. (*) The amount of the control cytokines IL-2, IFNγ, TNFα, and IL-10 produced in each transfection varies between 300 pg to 5 ng/ml.

Costimulation Assay.

A costimulation assay on resting PBL (peripheral blood lymphocytes) is performed in the presence of immobilized antibodies to CD3 and CD28. The use of antibodies specific for the invariant regions of CD3 mimic the induction of T cell activation that would occur through stimulation of the T cell receptor by an antigen. Cross-linking of the TCR (first signal) in the absence of a costimulatory signal (second signal) causes very low induction of proliferation and will eventually result in a state of “anergy”, which is characterized by the absence of growth and inability to produce cytokines. The addition of a costimulatory signal such as an antibody to CD28 mimics the action of the costimulatory molecule. B7-1 expressed on activated APCs, results in enhancement of T cell responses including cell survival and production of IL-2. Therefore this type of assay allows to detect both positive and negative effects caused by addition of supernatants expressing the proteins of interest on T cell proliferation.

The assay is performed as follows. Ninety-six well plates are coated with 100 ng/ml anti-CD3 and 5 μg/ml anti-CD28 (Pharmingen, San Diego, Calif.) in a final volume of 100 ul and incubated overnight at 4° C. Plates are washed twice with PBS before use. PBMC are isolated by FICOLL™ (LSM, ICN Biotechnologies, Aurora, Ohio) gradient centrifugation from human peripheral blood, and are cultured overnight in 10% FCS (Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight incubation period allows the adherent cells to attach to the plastic, which results in a lower background in the assay as there are fewer cells that can act as antigen presenting cells or that might be producing growth factors. The following day the non adherent cells are collected, washed and used in the proliferation assay. The assay is performed in a 96 well plate using 2×10⁴ cells/well in a final volume of 200 μl. The supernatants (e.g., CHO or 293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 60 ul are added to 140 μl of 10% FCS/RPMI containing the cells. Control supernatants are used at the same final dilution and express the following proteins: vector only (negative control), IL-2, IFNγ, TNFα, IL-10 and TR2. In addition to the control supernatants recombinant human IL-2 (R & D Systems, Minneapolis, Minn.) at a final concentration of 10 ng/ml is also used. After 24 hours of culture, each well is pulsed with 1 μCi of ³H-thymidine (Nen, Boston, Mass.). Cells are then harvested 20 hours following pulsing and incorporation of ³H-thymidine is used as a measure of proliferation. Results are expressed as an average of triplicate samples plus or minus standard error.

Costimulation Assay: IFNγ and IL-2 ELISA.

The assay is performed as follows. Twenty-four well plates are coated with either 300 ng/ml or 600 ng/ml anti-CD3 and 5 μg/ml anti-CD28 (Pharmingen, San Diego, Calif.) in a final volume of 500 ul and incubated overnight at 4° C. Plates are washed twice with PBS before use. PBMC are isolated by FICOLL™ (LSM, ICN Biotechnologies, Aurora, Ohio) gradient centrifugation from human peripheral blood, and are cultured overnight in 10% FCS (Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.). This overnight incubation period allows the adherent cells to attach to the plastic, which results in a lower background in the assay as there are fewer cells that can act as antigen presenting cells or that might be producing growth factors. The following day the non adherent cells are collected, washed and used in the costimulation assay. The assay is performed in the pre-coated twenty-four well plate using 1×10⁵ cells/well in a final volume of 900 μl. The supernatants (293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 300 μl are added to 600 μl of 10% FCS/RPMI containing the cells. Control supernatants are used at the same final dilution and express the following proteins: vector only (negative control), IL-2, IFNγ, IL-12 and IL-18. In addition to the control supernatants recombinant human IL-2 (all cytokines were purchased from R & D Systems, Minneapolis, Minn.) at a final concentration of 10 ng/ml, IL-12 at a final concentration of 1 ng/ml and IL-18 at a final concentration of 50 ng/ml are also used. Controls and unknown samples are tested in duplicate. Supernatant samples (250 μl) are collected 2 days and 5 days after the beginning of the assay. ELISAs to test for IFNγ and IL-2 secretion are performed using kits purchased from R & D Systems, (Minneapolis, Minn.). Results are expressed as an average of duplicate samples plus or minus standard error.

Proliferation Assay for Preactivated-Resting T Cells.

A proliferation assay on preactivated-resting T cells is performed on cells that are previously activated with the lectin phytohemagglutinin (PHA). Lectins are polymeric plant proteins that can bind to residues on T cell surface glycoproteins including the TCR and act as polyclonal activators. PBLs treated with PHA and then cultured in the presence of low doses of IL-2 resemble effector T cells. These cells are generally more sensitive to further activation induced by growth factors such as IL-2. This is due to the expression of high affinity IL-2 receptors that allows this population to respond to amounts of IL-2 that are 100 fold lower than what would have an effect on a naïve T cell. Therefore the use of this type of cells might enable to detect the effect of very low doses of an unknown growth factor, that would not be sufficient to induce proliferation on resting (naïve) T cells.

The assay is performed as follows. PBMC are isolated by F/H gradient centrifugation from human peripheral blood, and are cultured in 10% FCS (Fetal Calf Serum, Biofluids, Rockville, Md.)/RPMI (Gibco BRL, Gaithersburg, Md.) in the presence of 2 ug/ml PHA (SIGMA™, Saint Louis, Mo.) for three days. The cells are then washed in PBS and cultured in 10% FCS/RPMI in the presence of 5 ng/ml of human recombinant IL-2 (R & D Systems, Minneapolis, Minn.) for 3 days. The cells are washed and rested in starvation medium (1% FCS/RPMI) for 16 hours prior to the beginning of the proliferation assay. An aliquot of the cells is analyzed by FACS to determine the percentage of T cells (CD3 positive cells) present; this usually ranges between 93-97% depending on the donor. The assay is performed in a 96 well plate using 2×10⁴ cells/well in a final volume of 200 μl. The supernatants (e.g., CHO or 293T supernatants) expressing the protein of interest are tested at a 30% final dilution, therefore 60 μl are added to 140 μl of in 10% FCS/RPMI containing the cells. Control supernatants are used at the same final dilution and express the following proteins: vector (negative control), IL-2, IFNγ, TNFα, IL-10 and TR2. In addition to the control supernatants recombinant human IL-2 at a final concentration of 10 ng/ml is also used. After 24 hours of culture, each well is pulsed with 1 μCi of ³H-thymidine (Nen, Boston, Mass.). Cells are then harvested 20 hours following pulsing and incorporation of ³H-thymidine is used as a measure of proliferation. Results are expressed as an average of triplicate samples plus or minus standard error.

The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).

Example 23 Effect of Agonists or Antagonists of the Invention on the Expression of MHC Class II, Costimulatory and Adhesion Molecules and Cell Differentiation of Monocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferating precursors found in the peripheral blood: adherent PBMC or elutriated monocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). These dendritic cells have the characteristic phenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHC class II antigens). Treatment with activating factors, such as TNF-α, causes a rapid change in surface phenotype (increased expression of MHC class I and II, costimulatory and adhesion molecules, downregulation of FCγRII, upregulation of CD83). These changes correlate with increased antigen-presenting capacity and with functional maturation of the dendritic cells.

FACS analysis of surface antigens is performed as follows. Cells are treated 1-3 days with increasing concentrations of agonist or antagonist of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4° C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Effect on the Production of Cytokines.

Cytokines generated by dendritic cells, in particular IL-12, are important in the initiation of T-cell dependent immune responses. IL-12 strongly influences the development of Thl helper T-cell immune response, and induces cytotoxic T and NK cell function. An ELISA is used to measure the IL-12 release as follows. Dendritic cells (10⁶/ml) are treated with increasing concentrations of agonists or antagonists of the invention for 24 hours. LPS (100 ng/ml) is added to the cell culture as positive control. Supernatants from the cell cultures are then collected and analyzed for IL-12 content using commercial ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)). The standard protocols provided with the kits are used.

Effect on the Expression of MHC Class II, Costimulatory and Adhesion Molecules.

Three major families of cell surface antigens can be identified on monocytes: adhesion molecules, molecules involved in antigen presentation, and Fc receptor. Modulation of the expression of MHC class II antigens and other costimulatory molecules, such as B7 and ICAM-1, may result in changes in the antigen presenting capacity of monocytes and ability to induce T cell activation. Increased expression of Fc receptors may correlate with improved monocyte cytotoxic activity, cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows. Monocytes are treated 1-5 days with increasing concentrations of agonists or antagonists of the invention or LPS (positive control), washed with PBS containing 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20 dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30 minutes at 4 degrees C. After an additional wash, the labeled cells are analyzed by flow cytometry on a FACScan (Becton Dickinson).

Monocyte Activation and/or Increased Survival.

Assays for molecules that activate (or alternatively, inactivate) monocytes and/or increase monocyte survival (or alternatively, decrease monocyte survival) are known in the art and may routinely be applied to determine whether a molecule of the invention functions as an inhibitor or activator of monocytes. Agonists or antagonists of the invention can be screened using the three assays described below. For each of these assays, Peripheral blood mononuclear cells (PBMC) are purified from single donor leukopacks (American Red Cross, Baltimore, Md.) by centrifugation through a HISTOPAQUE™ gradient (SIGMA™). Monocytes are isolated from PBMC by counterflow centrifugal elutriation.

Monocyte Survival Assay.

Human peripheral blood monocytes progressively lose viability when cultured in absence of serum or other stimuli. Their death results from internally regulated processes (apoptosis). Addition to the culture of activating factors, such as TNF-alpha dramatically improves cell survival and prevents DNA fragmentation. Propidium iodide (PI) staining is used to measure apoptosis as follows. Monocytes are cultured for 48 hours in polypropylene tubes in serum-free medium (positive control), in the presence of 100 ng/ml TNF-alpha (negative control), and in the presence of varying concentrations of the compound to be tested. Cells are suspended at a concentration of 2×10⁶/ml in PBS containing PI at a final concentration of 5 μg/ml, and then incubated at room temperature for 5 minutes before FACScan analysis. PI uptake has been demonstrated to correlate with DNA fragmentation in this experimental paradigm.

Effect on Cytokine Release.

An important function of monocytes/macrophages is their regulatory activity on other cellular populations of the immune system through the release of cytokines after stimulation. An ELISA to measure cytokine release is performed as follows. Human monocytes are incubated at a density of 5×10⁵ cells/ml with increasing concentrations of agonists or antagonists of the invention and under the same conditions, but in the absence of agonists or antagonists. For IL-12 production, the cells are primed overnight with IFN (100 U/ml) in the presence of agonist or antagonist of the invention. LPS (10 ng/ml) is then added. Conditioned media are collected after 24 h and kept frozen until use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performed using a commercially available ELISA kit (e.g., R & D Systems (Minneapolis, Minn.)) and applying the standard protocols provided with the kit.

Oxidative Burst.

Purified monocytes are plated in 96-well plate at 2−1×10⁵ cell/well. Increasing concentrations of agonists or antagonists of the invention are added to the wells in a total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamine and antibiotics). After 3 days incubation, the plates are centrifuged and the medium is removed from the wells. To the macrophage monolayers, 0.2 ml per well of phenol red solution (140 mM NaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/ml of HRPO) is added, together with the stimulant (200 nM PMA). The plates are incubated at 37° C. for 2 hours and the reaction is stopped by adding 20 μl 1N NaOH per well. The absorbance is read at 610 nm. To calculate the amount of H₂O₂ produced by the macrophages, a standard curve of a H₂O₂ solution of known molarity is performed for each experiment.

The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).

Example 24 Biological Effects of Agonists or Antagonists of the Invention Astrocyte and Neuronal Assays.

Agonists or antagonists of the invention, expressed in Escherichia coli and purified as described above, can be tested for activity in promoting the survival, neurite outgrowth, or phenotypic differentiation of cortical neuronal cells and for inducing the proliferation of glial fibrillary acidic protein immunopositive cells, astrocytes. The selection of cortical cells for the bioassay is based on the prevalent expression of FGF-1 and FGF-2 in cortical structures and on the previously reported enhancement of cortical neuronal survival resulting from FGF-2 treatment. A thymidine incorporation assay, for example, can be used to elucidate an agonist or antagonist of the invention's activity on these cells.

Moreover, previous reports describing the biological effects of FGF-2 (basic FGF) on cortical or hippocampal neurons in vitro have demonstrated increases in both neuron survival and neurite outgrowth (Walicke et al., “Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension.” Proc. Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated by reference in its entirety). However, reports from experiments done on PC-12 cells suggest that these two responses are not necessarily synonymous and may depend on not only which FGF is being tested but also on which receptor(s) are expressed on the target cells. Using the primary cortical neuronal culture paradigm, the ability of an agonist or antagonist of the invention to induce neurite outgrowth can be compared to the response achieved with FGF-2 using, for example, a thymidine incorporation assay.

Fibroblast and Endothelial Cell Assays.

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.) and maintained in growth media from Clonetics. Dermal microvascular endothelial cells are obtained from Cell Applications (San Diego, Calif.). For proliferation assays, the human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells/well in a 96-well plate for one day in growth medium. The cells are then incubated for one day in 0.1% BSA basal medium. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test proteins for 3 days. ALAMAR BLUE™ (Alamar Biosciences, Sacramento, Calif.) is added to each well to a final concentration of 10%. The cells are incubated for 4 hr. Cell viability is measured by reading in a CYTOFLUOR™ fluorescence reader. For the PGE₂ assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or agonists or antagonists of the invention with or without IL-1α for 24 hours. The supernatants are collected and assayed for PGE₂ by EIA kit (Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lung fibroblasts are cultured at 5,000 cells/well in a 96-well plate for one day. After a medium change to 0.1% BSA basal medium, the cells are incubated with FGF-2 or with or without agonists or antagonists of the invention IL-1α for 24 hours. The supernatants are collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or agonists or antagonists of the invention for 3 days in basal medium before the addition of ALAMAR BLUE™ to assess effects on growth of the fibroblasts. FGF-2 should show a stimulation at 10-2500 ng/ml which can be used to compare stimulation with agonists or antagonists of the invention.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to a deficiency of striatal dopamine resulting from the degeneration of the nigrostriatal dopaminergic projection neurons. An animal model for Parkinson's that has been extensively characterized involves the systemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine (MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized by monoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP⁺) and released. Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons by the high-affinity reuptake transporter for dopamine. MPP⁺ is then concentrated in mitochondria by the electrochemical gradient and selectively inhibits nicotidamide adenine disphosphate: ubiquinone oxidoreductionase (complex I), thereby interfering with electron transport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basic FGF) has trophic activity towards nigral dopaminergic neurons (Ferrari et al., Dev. Biol. 1989). Recently, Dr. Unsicker's group has demonstrated that administering FGF-2 in gel foam implants in the striatum results in the near complete protection of nigral dopaminergic neurons from the toxicity associated with MPTP exposure (Otto and Unsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, agonists or antagonists of the invention can be evaluated to determine whether it has an action similar to that of FGF-2 in enhancing dopaminergic neuronal survival in vitro and it can also be tested in vivo for protection of dopaminergic neurons in the striatum from the damage associated with MPTP treatment. The potential effect of an agonist or antagonist of the invention is first examined in vitro in a dopaminergic neuronal cell culture paradigm. The cultures are prepared by dissecting the midbrain floor plate from gestation day 14 Wistar rat embryos. The tissue is dissociated with trypsin and seeded at a density of 200,000 cells/cm² on polyorthinine-laminin coated glass coverslips. The cells are maintained in Dulbecco's Modified Eagle's medium and F12 medium containing hormonal supplements (N1). The cultures are fixed with paraformaldehyde after 8 days in vitro and are processed for tyrosine hydroxylase, a specific marker for dopaminergic neurons, immunohistochemical staining. Dissociated cell cultures are prepared from embryonic rats. The culture medium is changed every third day and the factors are also added at that time.

Since the dopaminergic neurons are isolated from animals at gestation day 14, a developmental time which is past the stage when the dopaminergic precursor cells are proliferating, an increase in the number of tyrosine hydroxylase immunopositive neurons would represent an increase in the number of dopaminergic neurons surviving in vitro. Therefore, if an agonist or antagonist of the invention acts to prolong the survival of dopaminergic neurons, it would suggest that the agonist or antagonist may be involved in Parkinson's Disease.

The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).

Example 25 The Effect of Agonists or Antagonists of the Invention on the Growth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at 2−5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the medium is replaced with M199 containing 10% FBS, 8 units/ml heparin. An agonist or antagonist of the invention, and positive controls, such as VEGF and basic FGF (bFGF) are added, at varying concentrations. On days 4 and 6, the medium is replaced. On day 8, cell number is determined with a Coulter Counter.

An increase in the number of HUVEC cells indicates that the compound of the invention may proliferate vascular endothelial cells, while a decrease in the number of HUVEC cells indicates that the compound of the invention inhibits vascular endothelial cells.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).

Example 26 Stimulatory Effect of Polypeptides of the Invention on the Proliferation of Vascular Endothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetric MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) assay with the electron coupling reagent PMS (phenazine methosulfate) was performed (CellTiter 96 AQ, PROMEGA™). Cells are seeded in a 96-well plate (5,000 cells/well) in 0.1 mL serum-supplemented medium and are allowed to attach overnight. After serum-starvation for 12 hours in 0.5% FBS, conditions (bFGF, VEGF₁₆₅ or a polypeptide of the invention in 0.5% FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed to incubate for 1 hour at 37° C. before measuring the absorbance at 490 nm in an ELISA plate reader. Background absorbance from control wells (some media, no cells) is subtracted, and seven wells are performed in parallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518 (1994).

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 27 Inhibition of PDGF-Induced Vascular Smooth Muscle Cell Proliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent, quiescent cells grown on the 4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then, the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h, immunocytochemistry is performed by using BrdUrd Staining Kit (Zymed Laboratories). In brief, the cells are incubated with the biotinylated mouse anti-BrdUrd antibody at 4 degrees C. for 2 h after being exposed to denaturing solution and then incubated with the streptavidin-peroxidase and diaminobenzidine. After counterstaining with hematoxylin, the cells are mounted for microscopic examination, and the BrdUrd-positive cells are counted. The BrdUrd index is calculated as a percent of the BrdUrd-positive cells to the total cell number. In addition, the simultaneous detection of the BrdUrd staining (nucleus) and the FITC uptake (cytoplasm) is performed for individual cells by the concomitant use of bright field illumination and dark field-UV fluorescent illumination. See, Hayashida et al., J. Biol. Chem. 6:271(36):21985-21992 (1996).

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 28 Stimulation of Endothelial Migration

This example will be used to explore the possibility that a polypeptide of the invention may stimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 well microchemotaxis chamber (Neuroprobe Inc., Cabin John, Md.; Falk, W., et al., J. Immunological Methods 1980; 33:239-247). Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 μM (Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for at least 6 hours at room temperature and dried under sterile air. Test substances are diluted to appropriate concentrations in M199 supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of the final dilution is placed in the lower chamber of the modified Boyden apparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between lower and upper chamber, 2.5×10⁵ cells suspended in 50 ul M199 containing 1% FBS are seeded in the upper compartment. The apparatus is then incubated for 5 hours at 37° C. in a humidified chamber with 5% CO2 to allow cell migration. After the incubation period, the filter is removed and the upper side of the filter with the non-migrated cells is scraped with a rubber policeman. The filters are fixed with methanol and stained with a Giemsa solution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration is quantified by counting cells of three random high-power fields (40×) in each well, and all groups are performed in quadruplicate.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 29 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be a mediator of vascular endothelium relaxation. Thus, activity of a polypeptide of the invention can be assayed by determining nitric oxide production by endothelial cells in response to the polypeptide.

Nitric oxide is measured in 96-well plates of confluent microvascular endothelial cells after 24 hours starvation and a subsequent 4 hr exposure to various levels of a positive control (such as VEGF-1) and the polypeptide of the invention. Nitric oxide in the medium is determined by use of the Griess reagent to measure total nitrite after reduction of nitric oxide-derived nitrate by nitrate reductase. The effect of the polypeptide of the invention on nitric oxide release is examined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with a NO-specific polarographic electrode connected to a NO meter (Iso-NO, World Precision Instruments Inc.) (1049). Calibration of the NO elements is performed according to the following equation:

2KNO₂+2KI+2H₂SO₄62NO+I₂+2H₂O+2K₂SO₄

The standard calibration curve is obtained by adding graded concentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) into the calibration solution containing K₁ and H₂SO₄. The specificity of the Iso-NO electrode to NO is previously determined by measurement of NO from authentic NO gas (1050). The culture medium is removed and HUVECs are washed twice with Dulbecco's phosphate buffered saline. The cells are then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-well plates, and the cell plates are kept on a slide warmer (Lab Line Instruments Inc.) To maintain the temperature at 37° C. The NO sensor probe is inserted vertically into the wells, keeping the tip of the electrode 2 mm under the surface of the solution, before addition of the different conditions. S-nitroso acetyl penicillamin (SNAP) is used as a positive control. The amount of released NO is expressed as picomoles per 1×10⁶ endothelial cells. All values reported are means of four to six measurements in each group (number of cell culture wells). See, Leak et al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

The studies described in this example tested activity of polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 30 Effect of Polypeptides of the Invention on Cord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked by differentiation of endothelial cells. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from Cell Applications, Inc. as proliferating (passage 2) cells and are cultured in Cell Applications' CADMEC Growth Medium and used at passage 5. For the in vitro angiogenesis assay, the wells of a 48-well cell culture plate are coated with Cell Applications' Attachment Factor Medium (200 ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wells at 7,500 cells/well and cultured overnight in Growth Medium. The Growth Medium is then replaced with 300 mg Cell Applications' Chord Formation Medium containing control buffer or a polypeptide of the invention (0.1 to 100 ng/ml) and the cells are cultured for an additional 48 hr. The numbers and lengths of the capillary-like chords are quantitated through use of the Boeckeler VIA-170 video image analyzer. All assays are done in triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control. b-estradiol (1 ng/ml) is used as a negative control. The appropriate buffer (without protein) is also utilized as a control.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 31 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system to examine angiogenesis. Blood vessel formation on CAM is easily visible and quantifiable. The ability of polypeptides of the invention to stimulate angiogenesis in CAM can be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and the Japanese quail (Coturnix coturnix) are incubated at 37.8° C. and 80% humidity. Differentiated CAM of 16-day-old chick and 13-day-old quail embryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chick eggs. The embryos are checked for normal development and the eggs sealed with cellotape. They are further incubated until Day 13. THERMANOX™ coverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm in diameter. Sterile and salt-free growth factors are dissolved in distilled water and about 3.3 mg/5 ml are pipetted on the disks. After air-drying, the inverted disks are applied on CAM. After 3 days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed in 0.12M sodium cacodylate buffer. They are photographed with a stereo microscope [Wild M8] and embedded for semi- and ultrathin sectioning as described above. Controls are performed with carrier disks alone.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 32 Angiogenesis Assay Using a MATRIGEL™ Implant in Mouse

In vivo angiogenesis assay of a polypeptide of the invention measures the ability of an existing capillary network to form new vessels in an implanted capsule of murine extracellular matrix material (MATRIGEL™). The protein is mixed with the liquid MATRIGEL™ at 4 degree C. and the mixture is then injected subcutaneously in mice where it solidifies. After 7 days, the solid “plug” of MATRIGEL™ is removed and examined for the presence of new blood vessels. MATRIGEL™ is purchased from Becton Dickinson Labware/Collaborative Biomedical Products.

When thawed at 4 degree C. the MATRIGEL™ material is a liquid. The MATRIGEL™ is mixed with a polypeptide of the invention at 150 ng/ml at 4 degrees C. and drawn into cold 3 ml syringes. Female C57B1/6 mice approximately 8 weeks old are injected with the mixture of MATRIGEL™ and experimental protein at 2 sites at the midventral aspect of the abdomen (0.5 ml/site). After 7 days, the mice are sacrificed by cervical dislocation, the MATRIGEL™ plugs are removed and cleaned (i.e., all clinging membranes and fibrous tissue is removed). Replicate whole plugs are fixed in neutral buffered 10% formaldehyde, embedded in paraffin and used to produce sections for histological examination after staining with Masson's Trichrome. Cross sections from 3 different regions of each plug are processed. Selected sections are stained for the presence of vWF. The positive control for this assay is bovine basic FGF (150 ng/ml). MATRIGEL™ alone is used to determine basal levels of angiogenesis.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 33 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of polynucleotides and polypeptides of the invention on ischemia, a rabbit hindlimb ischemia model is created by surgical removal of one femoral artery as described previously (Takeshita et al., Am J. Pathol 147:1649-1660 (1995)). The excision of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. Consequently, blood flow to the ischemic limb is dependent upon collateral vessels originating from the internal iliac artery (Takeshita et al. Am J. Pathol 147:1649-1660 (1995)). An interval of 10 days is allowed for post-operative recovery of rabbits and development of endogenous collateral vessels. At 10 day post-operatively (day 0), after performing a baseline angiogram, the internal iliac artery of the ischemic limb is transfected with 500 mg naked expression plasmid containing a polynucleotide of the invention by arterial gene transfer technology using a hydrogel-coated balloon catheter as described (Riessen et al. Hum Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin. Invest. 90: 936-944 (1992)). When a polypeptide of the invention is used in the treatment, a single bolus of 500 mg polypeptide of the invention or control is delivered into the internal iliac artery of the ischemic limb over a period of 1 min. through an infusion catheter. On day 30, various parameters are measured in these rabbits: (a) BP ratio—The blood pressure ratio of systolic pressure of the ischemic limb to that of normal limb; (b) Blood Flow and Flow Reserve—Resting FL: the blood flow during undilated condition and Max FL: the blood flow during fully dilated condition (also an indirect measure of the blood vessel amount) and Flow Reserve is reflected by the ratio of max FL:resting FL; (c) Angiographic Score—This is measured by the angiogram of collateral vessels. A score is determined by the percentage of circles in an overlaying grid that with crossing opacified arteries divided by the total number m the rabbit thigh; (d) Capillary density—The number of collateral capillaries determined in light microscopic sections taken from hindlimbs.

The studies described in this example tested activity of polynucleotides and polypeptides of the invention. However, one skilled in the art could easily modify the exemplified studies to test the agonists, and/or antagonists of the invention.

Example 34 Effect of Polypeptides of the Invention on Vasodilation

Since dilation of vascular endothelium is important in reducing blood pressure, the ability of polypeptides of the invention to affect the blood pressure in spontaneously hypertensive rats (SHR) is examined. Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of the polypeptides of the invention are administered to 13-14 week old spontaneously hypertensive rats (SHR). Data are expressed as the mean+/−SEM. Statistical analysis are performed with a paired t-test and statistical significance is defined as p<0.05 vs. the response to buffer alone.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 35 Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction. Expression of polypeptides of the invention, during the skin ischemia, is studied using in situ hybridization.

The study in this model is divided into three parts as follows:

-   -   Ischemic skin     -   Ischemic skin wounds     -   Normal wounds

The experimental protocol includes:

-   -   Raising a 3×4 cm, single pedicle full-thickness random skin flap         (myocutaneous flap over the lower back of the animal).     -   An excisional wounding (4-6 mm in diameter) in the ischemic skin         (skin-flap).     -   Topical treatment with a polypeptide of the invention of the         excisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the         following various dosage ranges: 1 mg to 100 mg.     -   Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21         post-wounding for histological, immunohistochemical, and in situ         studies.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 36 Peripheral Arterial Disease Model

Angiogenic therapy using a polypeptide of the invention is a novel therapeutic strategy to obtain restoration of blood flow around the ischemia in case of peripheral arterial diseases. The experimental protocol includes:

One side of the femoral artery is ligated to create ischemic muscle of the hindlimb, the other side of hindlimb serves as a control.

A polypeptide of the invention, in a dosage range of 20 mg-500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-3 weeks.

The ischemic muscle tissue is collected after ligation of the femoral artery at 1, 2, and 3 weeks for the analysis of expression of a polypeptide of the invention and histology. Biopsy is also performed on the other side of normal muscle of the contralateral hindlimb.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 37 Ischemic Myocardial Disease Model

A polypeptide of the invention is evaluated as a potent mitogen capable of stimulating the development of collateral vessels, and restructuring new vessels after coronary artery occlusion. Alteration of expression of the polypeptide is investigated in situ. The experimental protocol includes:

The heart is exposed through a left-side thoracotomy in the rat. Immediately, the left coronary artery is occluded with a thin suture (6-0) and the thorax is closed.

A polypeptide of the invention, in a dosage range of 20 mg-500 mg, is delivered intravenously and/or intramuscularly 3 times (perhaps more) per week for 2-4 weeks.

Thirty days after the surgery, the heart is removed and cross-sectioned for morphometric and in situ analyzes.

The studies described in this example tested activity of a polypeptide of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), agonists, and/or antagonists of the invention.

Example 38 Rat Corneal Wound Healing Model

This animal model shows the effect of an agonist or antagonist of the invention on neovascularization. The experimental protocol includes:

a) Making a 1-1.5 mm long incision from the center of cornea into the stromal layer. b) Inserting a spatula below the lip of the incision facing the outer corner of the eye. c) Making a pocket (its base is 1-1.5 mm form the edge of the eye). d) Positioning a pellet, containing 50 ng-5 μg of an agonist or antagonist of the invention, within the pocket. e) Treatment with an agonist or antagonist of the invention can also be applied topically to the corneal wounds in a dosage range of 20 mg-500 mg (daily treatment for five days).

The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).

Example 39 Diabetic Mouse and Glucocorticoid-Impaired Wound Healing Models

Diabetic db+/db+ Mouse Model.

To demonstrate that an agonist or antagonist of the invention accelerates the healing process, the genetically diabetic mouse model of wound healing is used. The full thickness wound healing model in the db+/db+ mouse is a well characterized, clinically relevant and reproducible model of impaired wound healing. Healing of the diabetic wound is dependent on formation of granulation tissue and re-epithelialization rather than contraction (Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observed in Type II diabetes mellitus. Homozygous (db+/db+) mice are obese in comparison to their normal heterozygous (db+/+m) littermates. Mutant diabetic (db+/db+) mice have a single autosomal recessive mutation on chromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293 (1982)). Animals show polyphagia, polydipsia and polyuria. Mutant diabetic mice (db+/db+) have elevated blood glucose, increased or normal insulin levels, and suppressed cell-mediated immunity (Mandel et al., J. Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol. 51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular lesions, basement membrane thickening and glomerular filtration abnormalities have been described in these animals (Norido, F. et al., Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes 29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979); Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)). These homozygous diabetic mice develop hyperglycemia that is resistant to insulin analogous to human type II diabetes (Mandel et al., J. Immunol. 120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing in this model may be similar to the healing observed in human diabetes (Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and their non-diabetic (db+/+m) heterozygous littermates are used in this study (Jackson Laboratories). The animals are purchased at 6 weeks of age and are 8 weeks old at the beginning of the study. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. The experiments are conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods (Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)). Briefly, on the day of wounding, animals are anesthetized with an intraperitoneal injection of Avertin™ (0.01 mg/mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water. The dorsal region of the animal is shaved and the skin washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is then created using a Keyes tissue punch. Immediately following wounding, the surrounding skin is gently stretched to eliminate wound expansion. The wounds are left open for the duration of the experiment. Application of the treatment is given topically for 5 consecutive days commencing on the day of wounding. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at the day of surgery and at two day intervals thereafter. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

An agonist or antagonist of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls) are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3) treated group.

Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total square area of the wound. Contraction is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm², the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds are used to assess whether the healing process and the morphologic appearance of the repaired skin is altered by treatment with an agonist or antagonist of the invention. This assessment included verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D. G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometer is used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonal rabbit anti-human keratin antibody using ABC Elite detection system. Human skin is used as a positive tissue control while non-immune IgG is used as a negative control. Keratinocyte growth is determined by evaluating the extent of reepithelialization of the wound using a calibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens is demonstrated by using anti-PCNA antibody (1:50) with an ABC Elite detection system. Human colon cancer served as a positive tissue control and human brain tissue is used as a negative tissue control. Each specimen included a section with omission of the primary antibody and substitution with non-immune mouse IgG. Ranking of these sections is based on the extent of proliferation on a scale of 0-8, the lower side of the scale reflecting slight proliferation to the higher side reflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.

Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, Glucocorticoids and Wound healing. In: Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahl et al., J. Immunol. 115: 476-481 (1975); Werb et al., J. Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retard wound healing by inhibiting angiogenesis, decreasing vascular permeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978)) and producing a transient reduction of circulating monocytes (Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989)). The systemic administration of steroids to impair wound healing is a well establish phenomenon in rats (Beck et al., Growth Factors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl, “Glucocorticoids and wound healing”, In: Antiinflammatory Steroid Action: Basic and Clinical Aspects, Academic Press, New York, pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA 86: 2229-2233 (1989)).

To demonstrate that an agonist or antagonist of the invention can accelerate the healing process, the effects of multiple topical applications of the agonist or antagonist on full thickness excisional skin wounds in rats in which healing has been impaired by the systemic administration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles River Laboratories) are used in this example. The animals are purchased at 8 weeks of age and are 9 weeks old at the beginning of the study. The healing response of rats is impaired by the systemic administration of methylprednisolone (17 mg/kg/rat intramuscularly) at the time of wounding. Animals are individually housed and received food and water ad libitum. All manipulations are performed using aseptic techniques. This study is conducted according to the rules and guidelines of Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On the day of wounding, animals are anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsal region of the animal is shaved and the skin washed with 70% ethanol and iodine solutions. The surgical area is dried with sterile gauze prior to wounding. An 8 mm full-thickness wound is created using a Keyes tissue punch. The wounds are left open for the duration of the experiment. Applications of the testing materials are given topically once a day for 7 consecutive days commencing on the day of wounding and subsequent to methylprednisolone administration. Prior to treatment, wounds are gently cleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at the day of wounding and at the end of treatment. Wound closure is determined by daily measurement on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. Wounds are considered healed if granulation tissue is no longer visible and the wound is covered by a continuous epithelium.

The agonist or antagonist of the invention is administered using at a range different doses, from 4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg/kg). The wounds and surrounding skin are then harvested for histology. Tissue specimens are placed in 10% neutral buffered formalin in tissue cassettes between biopsy sponges for further processing.

Three groups of 10 animals each (5 with methylprednisolone and 5 without glucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebo control 3) treated groups.

Wound closure is analyzed by measuring the area in the vertical and horizontal axis and obtaining the total area of the wound. Closure is then estimated by establishing the differences between the initial wound area (day 0) and that of post treatment (day 8). The wound area on day 1 is 64 mm², the corresponding size of the dermal punch. Calculations are made using the following formula:

[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embedded blocks are sectioned perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H&E) staining is performed on cross-sections of bisected wounds. Histologic examination of the wounds allows assessment of whether the healing process and the morphologic appearance of the repaired skin is improved by treatment with an agonist or antagonist of the invention. A calibrated lens micrometer is used by a blinded observer to determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of <0.05 is considered significant.

The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).

Example 40 Lymphadema Animal Model

The purpose of this experimental approach is to create an appropriate and consistent lymphedema model for testing the therapeutic effects of an agonist or antagonist of the invention in lymphangiogenesis and re-establishment of the lymphatic circulatory system in the rat hind limb. Effectiveness is measured by swelling volume of the affected limb, quantification of the amount of lymphatic vasculature, total blood plasma protein, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progress of the edema is followed for up to 3-4 weeks.

Prior to beginning surgery, blood sample is drawn for protein concentration analysis. Male rats weighing approximately ˜350 g are dosed with Pentobarbital. Subsequently, the right legs are shaved from knee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. Circumference and volumetric measurements are made prior to injecting dye into paws after marking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsal paw). The intradermal dorsum of both right and left paws are injected with 0.05 ml of 1% Evan's Blue. Circumference and volumetric measurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is made circumferentially allowing the femoral vessels to be located. Forceps and hemostats are used to dissect and separate the skin flaps. After locating the femoral vessels, the lymphatic vessel that runs along side and underneath the vessel(s) is located. The main lymphatic vessels in this area are then electrically coagulated or suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosis and adductors) are bluntly dissected. The popliteal lymph node is then located. The 2 proximal and 2 distal lymphatic vessels and distal blood supply of the popliteal node are then ligated by suturing. The popliteal lymph node, and any accompanying adipose tissue, is then removed by cutting connective tissues.

Care is taken to control any mild bleeding resulting from this procedure. After lymphatics are occluded, the skin flaps are sealed by using liquid skin (Vetbond) (A J Buck). The separated skin edges are sealed to the underlying muscle tissue while leaving a gap of ˜0.5 cm around the leg. Skin also may be anchored by suturing to underlying muscle when necessary.

To avoid infection, animals are housed individually with mesh (no bedding). Recovering animals are checked daily through the optimal edematous peak, which typically occurred by day 5-7. The plateau edematous peak are then observed. To evaluate the intensity of the lymphedema, the circumference and volumes of 2 designated places on each paw before operation and daily for 7 days are measured. The effect of plasma proteins on lymphedema is determined and whether protein analysis is a useful testing perimeter is also investigated. The weights of both control and edematous limbs are evaluated at 2 places. Analysis is performed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limb movement, a cloth tape is used to measure limb circumference. Measurements are done at the ankle bone and dorsal paw by 2 different people and those 2 readings are averaged. Readings are taken from both control and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetized with Pentobarbital and are tested prior to surgery. For daily volumetrics animals are under brief halothane anesthetic (rapid immobilization and quick recovery), and both legs are shaved and equally marked using waterproof marker on legs. Legs are first dipped in water, then dipped into the instrument to each marked level then measured by Buxco edema software (Chen/Victor). Data is recorded by one person, while the other is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serum separated prior to surgery and then at conclusion for total protein and Ca2⁺ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared for tissue collection. The limbs are amputated using a quillitine, then both experimental and control legs are cut at the ligature and weighed. A second weighing is done as the tibio-cacaneal joint is disarticulated and the foot is weighed.

Histological Preparations: The transverse muscle located behind the knee (popliteal) area is dissected and arranged in a metal mold, filled with freezeGel, dipped into cold methylbutane, placed into labeled sample bags at −80 EC until sectioning. Upon sectioning, the muscle is observed under fluorescent microscopy for lymphatics.

The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).

Example 41 Suppression of TNF Alpha-Induced Adhesion Molecule Expression by an Agonist or Antagonist of the Invention

The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.

Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine, is a stimulator of all three CAMs on endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in a pathological outcome.

The potential of an agonist or antagonist of the invention to mediate a suppression of TNF-a induced CAM expression can be examined. A modified ELISA assay which uses ECs as a solid phase absorbent is employed to measure the amount of CAM expression on TNF-a treated ECs when co-stimulated with a member of the FGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC) cultures are obtained from pooled cord harvests and maintained in growth medium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCS and 1% penicillin/streptomycin in a 37 degree C. humidified incubator containing 5% CO₂. HUVECs are seeded in 96-well plates at concentrations of 1×10⁴ cells/well in EGM medium at 37 degree C. for 18-24 hrs or until confluent. The monolayers are subsequently washed 3 times with a serum-free solution of RPMI-1640 supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin, and treated with a given cytokine and/or growth factor(s) for 24 h at 37 degree C. Following incubation, the cells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard 96 well plate to confluence. Growth medium is removed from the cells and replaced with 90 μl of 199 Medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 μl volumes). Plates are incubated at 37 degree C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min.

Fixative is then removed from the wells and wells are washed 1× with PBS(+Ca,Mg)+0.5% BSA and drained. The wells should not be allowed to dry. 10 μl of diluted primary antibody is then added to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.

Then add 20 μl of diluted Extravidin®-Alkaline Phosphotase (1:5,000 dilution) to each well and incubated at 37° C. for 30 min. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol Phosphate pNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the Extravidin®-Alkaline Phosphotase in glycine buffer: 1:5,000 (10⁰)>10^(−0.5)>10⁻¹>10^(−1.5)0.5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added to each of the standard wells. The plate must be incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on blank wells filled with glycine buffer only. The template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.

The studies described in this example tested activity of agonists or antagonists of the invention. However, one skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides or polypeptides of the invention (e.g., gene therapy).

Example 42 Production of Polypeptide of the Invention for High-Throughput Screening Assays

The following protocol produces a supernatant containing polypeptide of the present invention to be tested. This supernatant can then be used in the Screening Assays described in Examples 44-53.

First, dilute Poly-D-Lysine (644 587 BOEHRINGER™ Mannheim) stock solution (1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker) for a working solution of 50 ug/ml. Add 200 μl of this solution to each well (24 well plates) and incubate at RT for 20 minutes. Be sure to distribute the solution over each well (note: a 12-channel pipetter may be used with tips on every other channel). Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS (Phosphate Buffered Saline). The PBS should remain in the well until just prior to plating the cells and plates may be poly-lysine coated in advance for up to two weeks.

Plate 293T cells (do not carry cells past P+20) at 2×10⁵ cells/well in 0.5 ml DMEM (Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose and L-glutamine (12-604F Biowhittaker))/10% heat inactivated FBS (14-503F Biowhittaker)/1× Penstrep (17-602E Biowhittaker). Let the cells grow overnight.

The next day, mix together in a sterile solution basin: 300 μl LIPOFECTAMINE™ (18324-012 Gibco/BRL) and 5 ml OPTI-MEM™ I (31985070 Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter, aliquot approximately 2 μg of an expression vector containing a polynucleotide insert, produced by the methods described in Examples 8-10, into an appropriately labeled 96-well round bottom plate. With a multi-channel pipetter, add 50 μl of the LIPOFECTAMINE™/OPTI-MEM™ I mixture to each well. Pipette up and down gently to mix. Incubate at RT 15-45 minutes. After about 20 minutes, use a multi-channel pipetter to add 150 μl OPTI-MEM™ I to each well. As a control, one plate of vector DNA lacking an insert should be transfected with each set of transfections.

Preferably, the transfection should be performed by tag-teaming the following tasks. By tag-teaming, hands on time is cut in half, and the cells do not spend too much time on PBS. First, person A aspirates off the media from four 24-well plates of cells, and then person B rinses each well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, and person B, using a12-channel pipetter with tips on every other channel, adds the 200 μl of DNA/LIPOFECTAMINE™/OPTI-MEM™ I complex to the odd wells first, then to the even wells, to each row on the 24-well plates. Incubate at 37 degree C. for 6 hours.

While cells are incubating, prepare appropriate media, either 1% BSA in DMEM with 1× penstrep, or HGS CHO-5 media (116.6 mg/L of CaCl2 (anhyd); 0.00130 mg/L CuSO₄-5H₂O; 0.050 mg/L of Fe(NO₃)₃-9H₂O; 0.417 mg/L of FeSO₄-7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/L of MgSO₄; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO₃; 62.50 mg/L of NaH₂PO₄—H₂O; 71.02 mg/L of Na₂HPO4; 0.4320 mg/L of ZnSO₄-7H₂O; 0.002 mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L of DL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L of Linolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid; 0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L of Pluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551 mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml of L-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂O; 6.65 mg/ml of L-Aspartic Acid; 29.56 mg/ml of L-Cystine-2HCL-H₂O; 31.29 mg/ml of L-Cystine-2HCL; 7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/ml of Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂O; 106.97 mg/ml of L-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL; 32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/ml of L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine; 19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂O; and 99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-Ca Pantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid; 15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L of Pyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin; 3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L of Vitamin B₁₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105 mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L of Sodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 μM of Ethanolamine; 0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrin complexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrin complexed with Oleic Acid; 10 mg/L of Methyl-B-Cyclodextrin complexed with Retinal Acetate. Adjust osmolarity to 327 mOsm) with 2 mm glutamine and 1×penstrep. (BSA (81-068-3 BAYER™) 100 gm dissolved in 1L DMEM for a 10% BSA stock solution). Filter the media and collect 50 μl for endotoxin assay in 15 ml polystyrene conical.

The transfection reaction is terminated, preferably by tag-teaming, at the end of the incubation period. Person A aspirates off the transfection media, while person B adds 1.5 ml appropriate media to each well. Incubate at 37 degree C. for 45 or 72 hours depending on the media used: 1% BSA for 45 hours or CHO-5 for 72 hours.

On day four, using a 300 μl multichannel pipetter, aliquot 600 μl in one 1 ml deep well plate and the remaining supernatant into a 2 ml deep well. The supernatants from each well can then be used in the assays described in Examples 44-51.

It is specifically understood that when activity is obtained in any of the assays described below using a supernatant, the activity originates from either the polypeptide of the present invention directly (e.g., as a secreted protein) or by polypeptide of the present invention inducing expression of other proteins, which are then secreted into the supernatant. Thus, the invention further provides a method of identifying the protein in the supernatant characterized by an activity in a particular assay.

Example 43 Construction of GAS Reporter Construct

One signal transduction pathway involved in the differentiation and proliferation of cells is called the Jaks-STATs pathway. Activated proteins in the Jaks-STATs pathway bind to gamma activation site “GAS” elements or interferon-sensitive responsive element (“ISRE”), located in the promoter of many genes. The binding of a protein to these elements alter the expression of the associated gene.

GAS and ISRE elements are recognized by a class of transcription factors called Signal Transducers and Activators of Transcription, or “STATs.” There are six members of the STATs family. Stat1 and Stat3 are present in many cell types, as is Stat2 (as response to IFN-alpha is widespread). Stat4 is more restricted and is not in many cell types though it has been found in T helper class I, cells after treatment with IL-12. Stat5 was originally called mammary growth factor, but has been found at higher concentrations in other cells including myeloid cells. It can be activated in tissue culture cells by many cytokines.

The STATs are activated to translocate from the cytoplasm to the nucleus upon tyrosine phosphorylation by a set of kinases known as the Janus Kinase (“Jaks”) family. Jaks represent a distinct family of soluble tyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinases display significant sequence similarity and are generally catalytically inactive in resting cells.

The Jaks are activated by a wide range of receptors summarized in the Table below. (Adapted from review by Schidler and Darnell, Ann. Rev. Biochem. 64:621-51 (1995)). A cytokine receptor family, capable of activating Jaks, is divided into two groups: (a) Class 1 includes receptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15, Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b) Class 2 includes IFN-α, IFN-g, and IL-10. The Class 1 receptors share a conserved cysteine motif (a set of four conserved cysteines and one tryptophan) and a WSXWS motif (a membrane proximal region encoding Trp-Ser-Xaa-Trp-Ser (SEQ ID NO:237)).

Thus, on binding of a ligand to a receptor, Jaks are activated, which in turn activate STATs, which then translocate and bind to GAS elements. This entire process is encompassed in the Jaks-STATs signal transduction pathway. Therefore, activation of the Jaks-STATs pathway, reflected by the binding of the GAS or the ISRE element, can be used to indicate proteins involved in the proliferation and differentiation of cells. For example, growth factors and cytokines are known to activate the Jaks-STATs pathway (See Table below). Thus, by using GAS elements linked to reporter molecules, activators of the Jaks-STATs pathway can be identified.

JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS(elements) or ISRE IFN family IFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 > IFP) Il-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotropic) + + + ? 1, 3 GAS (IRF1 > Lys6 > IFP) Il-11(Pleiotropic) ? + ? ? 1, 3 OnM(Pleiotropic) ? + + ? 1, 3 LIF(Pleiotropic) ? + + ? 1, 3 CNTF(Pleiotropic) −/+ + + ? 1, 3 G-CSF(Pleiotropic) ? + ? ? 1, 3 IL-12(Pleiotropic) + − + + 1, 3 g-C family IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid) − + − + 6 GAS (IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GAS IL-9 (lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15 ? + ? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >> Ly6) IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growth hormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5 GAS (B-CAS > IRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1, 3 GAS (IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

To construct a synthetic GAS containing promoter element, which is used in the Biological Assays described in Examples 44-45, a PCR based strategy is employed to generate a GAS-SV40 promoter sequence. The 5′ primer contains four tandem copies of the GAS binding site found in the IRF1 promoter and previously demonstrated to bind STATs upon induction with a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).), although other GAS or ISRE elements can be used instead. The 5′ primer also contains 18 bp of sequence complementary to the SV40 early promoter sequence and is flanked with an XhoI site. The sequence of the 5′ primer is: 5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGAT TTCCCCGAAATATCTGCCATCTCAATTAG:3′ (SEQ ID NO:238)

The downstream primer is complementary to the SV40 promoter and is flanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO:239)

PCR amplification is performed using the SV40 promoter template present in the B-gal:promoter plasmid obtained from CLONTECH™. The resulting PCR fragment is digested with XhoI/Hind III and subcloned into BLSK2-. (STRATAGENE™.) Sequencing with forward and reverse primers confirms that the insert contains the following sequence: 5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCC CGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCAT CCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTT TATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGA GGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′ (SEQ ID NO:240)

With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2 reporter construct is next engineered. Here, the reporter molecule is a secreted alkaline phosphatase, or “SEAP.” Clearly, however, any reporter molecule can be instead of SEAP, in this or in any of the other Examples. Well known reporter molecules that can be used instead of SEAP include chloramphenicol acetyltransferase (CAT), luciferase, alkaline phosphatase, B-galactosidase, green fluorescent protein (GFP), or any protein detectable by an antibody.

The above sequence confirmed synthetic GAS-SV40 promoter element is subcloned into the pSEAP-Promoter vector obtained from CLONTECH™ using HindIII and XhoI, effectively replacing the SV40 promoter with the amplified GAS:SV40 promoter element, to create the GAS-SEAP vector. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.

Thus, in order to generate mammalian stable cell lines expressing the GAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAP vector using SalI and NotI, and inserted into a backbone vector containing the neomycin resistance gene, such as pGFP-1 (CLONTECH™), using these restriction sites in the multiple cloning site, to create the GAS-SEAP/Neo vector. Once this vector is transfected into mammalian cells, this vector can then be used as a reporter molecule for GAS binding as described in Examples 44-45.

Other constructs can be made using the above description and replacing GAS with a different promoter sequence. For example, construction of reporter molecules containing EGR and NF-KB promoter sequences are described in Examples 46 and 47. However, many other promoters can be substituted using the protocols described in these Examples. For instance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted, alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, I1-2/NFAT, or NF-KB/GAS). Similarly, other cell lines can be used to test reporter construct activity, such as HELA (epithelial), HUVEC (endothelial), Reh (B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.

Example 44 High-Throughput Screening Assay for T-Cell Activity

The following protocol is used to assess T-cell activity by identifying factors, and determining whether supernatant containing a polypeptide of the invention proliferates and/or differentiates T-cells. T-cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 43. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The T-cell used in this assay is Jurkat T-cells (ATCC™ Accession No. TIB-152), although Molt-3 cells (ATCC™ Accession No. CRL-1552) and Molt-4 cells (ATCC™ Accession No. CRL-1582) cells can also be used.

Jurkat T-cells are lymphoblastic CD4+ Thl helper cells. In order to generate stable cell lines, approximately 2 million Jurkat cells are transfected with the GAS-SEAP/neo vector using DMRIE-C (LIFE TECHNOLOGIES™)(transfection procedure described below). The transfected cells are seeded to a density of approximately 20,000 cells per well and transfectants resistant to 1 mg/ml genticin selected. Resistant colonies are expanded and then tested for their response to increasing concentrations of interferon gamma. The dose response of a selected clone is demonstrated.

Specifically, the following protocol will yield sufficient cells for 75 wells containing 200 μl of cells. Thus, it is either scaled up, or performed in multiple to generate sufficient cells for multiple 96 well plates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep. Combine 2.5 mls of OPTI-MEM™ (LIFE TECHNOLOGIES™) with 10 μg of plasmid DNA in a T25 flask. Add 2.5 ml OPTI-MEM™ containing 50 μl of DMRIE-C and incubate at room temperature for 15-45 mins.

During the incubation period, count cell concentration, spin down the required number of cells (10⁷ per transfection), and resuspend in OPTI-MEM™ to a final concentration of 10⁷ cells/ml. Then add 1 ml of 1×10⁷ cells in OPTI-MEM™ to T25 flask and incubate at 37 degree C. for 6 hrs. After the incubation, add 10 ml of RPMI+15% serum.

The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10% serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated with supernatants containing polypeptide of the present invention or polypeptide of the present invention induced polypeptides as produced by the protocol described in Example 42.

On the day of treatment with the supernatant, the cells should be washed and resuspended in fresh RPMI+10% serum to a density of 500,000 cells per ml. The exact number of cells required will depend on the number of supernatants being screened. For one 96 well plate, approximately 10 million cells (for 10 plates, 100 million cells) are required.

Transfer the cells to a triangular reservoir boat, in order to dispense the cells into a 96 well dish, using a 12 channel pipette. Using a 12 channel pipette, transfer 200 μl of cells into each well (therefore adding 100,000 cells per well).

After all the plates have been seeded, 50 μl of the supernatants are transferred directly from the 96 well plate containing the supernatants into each well using a 12 channel pipette. In addition, a dose of exogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10, and H11 to serve as additional positive controls for the assay.

The 96 well dishes containing Jurkat cells treated with supernatants are placed in an incubator for 48 hrs (note: this time is variable between 48-72 hrs). 35 μl samples from each well are then transferred to an opaque 96 well plate using a 12 channel pipette. The opaque plates should be covered (using sellophene covers) and stored at −20 degree C. until SEAP assays are performed according to Example 48. The plates containing the remaining treated cells are placed at 4 degree C. and serve as a source of material for repeating the assay on a specific well if desired.

As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate Jurkat T cells. Over 30 fold induction is typically observed in the positive control wells.

The above protocol may be used in the generation of both transient, as well as, stable transfected cells, which would be apparent to those of skill in the art.

Example 45 High-Throughput Screening Assay Identifying Myeloid Activity

The following protocol is used to assess myeloid activity of polypeptide of the present invention by determining whether polypeptide of the present invention proliferates and/or differentiates myeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neo construct produced in Example 43. Thus, factors that increase SEAP activity indicate the ability to activate the Jaks-STATS signal transduction pathway. The myeloid cell used in this assay is U937, a pre-monocyte cell line, although TF-1, HL60, or KG1 can be used.

To transiently transfect U937 cells with the GAS/SEAP/Neo construct produced in Example 43, a DEAE-Dextran method (Kharbanda et. al., 1994, Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×10⁷ U937 cells and wash with PBS. The U937 cells are usually grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.

Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffer containing 0.5 mg/ml DEAE-Dextran, 8 μg GAS-SEAP2 plasmid DNA, 140 mM NaCl, 5 mM KCl, 375 μM Na₂HPO₄.7H₂O, 1 mM MgCl₂, and 675 μM CaCl₂. Incubate at 37 degrees C. for 45 min.

Wash the cells with RPMI 1640 medium containing 10% FBS and then resuspend in 10 ml complete medium and incubate at 37 degree C. for 36 hr.

The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400 μg/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 400 μg/ml G418 for couple of passages.

These cells are tested by harvesting 1×10⁸ cells (this is enough for ten 96-well plates assay) and wash with PBS. Suspend the cells in 200 ml above described growth medium, with a final density of 5×10⁵ cells/ml. Plate 200 μl cells per well in the 96-well plate (or 1×10⁵ cells/well).

Add 50 μl of the supernatant prepared by the protocol described in Example 42. Incubate at 37 degree C. for 48 to 72 hr. As a positive control, 100 Unit/ml interferon gamma can be used which is known to activate U937 cells. Over 30 fold induction is typically observed in the positive control wells. SEAP assay the supernatant according to the protocol described in Example 48.

Example 46 High-Throughput Screening Assay Identifying Neuronal Activity

When cells undergo differentiation and proliferation, a group of genes are activated through many different signal transduction pathways. One of these genes, EGR1 (early growth response gene 1), is induced in various tissues and cell types upon activation. The promoter of EGR1 is responsible for such induction. Using the EGR1 promoter linked to reporter molecules, activation of cells can be assessed by polypeptide of the present invention.

Particularly, the following protocol is used to assess neuronal activity in PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are known to proliferate and/or differentiate by activation with a number of mitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growth factor), and EGF (epidermal growth factor). The EGR1 gene expression is activated during this treatment. Thus, by stably transfecting PC12 cells with a construct containing an EGR promoter linked to SEAP reporter, activation of PC12 cells by polypeptide of the present invention can be assessed.

The EGR/SEAP reporter construct can be assembled by the following protocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al., Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNA using the following primers: 5′ GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ (SEQ ID NO:241) 5′ GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′ (SEQ ID NO:242)

Using the GAS:SEAP/Neo vector produced in Example 43, EGR1 amplified product can then be inserted into this vector. Linearize the GAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing the GAS/SV40 stuffer. Restrict the EGR1 amplified product with these same enzymes. Ligate the vector and the EGR1 promoter.

To prepare 96 well-plates for cell culture, two mls of a coating solution (1:30 dilution of collagen type I (Upstate Biotech Inc. Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cm plate or 50 ml per well of the 96-well plate, and allowed to air dry for 2 hr.

PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker) containing 10% horse serum (JRH BIOSCIENCES, Cat. #12449-78P), 5% heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin on a precoated 10 cm tissue culture dish. One to four split is done every three to four days. Cells are removed from the plates by scraping and resuspended with pipetting up and down for more than 15 times.

Transfect the EGR/SEAP/Neo construct into PC12 using the LIPOFECTAMINE™ protocol described in Example 42. EGR-SEAP/PC12 stable cells are obtained by growing the cells in 300 μg/ml G418. The G418-free medium is used for routine growth but every one to two months, the cells should be re-grown in 300 μg/ml G418 for couple of passages.

To assay for neuronal activity, a 10 cm plate with cells around 70 to 80% confluent is screened by removing the old medium. Wash the cells once with PBS (Phosphate buffered saline). Then starve the cells in low serum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS with antibiotics) overnight.

The next morning, remove the medium and wash the cells with PBS. Scrape off the cells from the plate, suspend the cells well in 2 ml low serum medium. Count the cell number and add more low serum medium to reach final cell density as 5×10⁵ cells/ml.

Add 200 μl of the cell suspension to each well of 96-well plate (equivalent to 1×10⁵ cells/well). Add 50 μl supernatant produced by Example 42, 37 degree C. for 48 to 72 hr. As a positive control, a growth factor known to activate PC12 cells through EGR can be used, such as 50 ng/μl of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAP is typically seen in the positive control wells. SEAP assay the supernatant according to Example 48.

Example 47 High-Throughput Screening Assay for T-Cell Activity

NF-KB (Nuclear Factor KB) is a transcription factor activated by a wide variety of agents including the inflammatory cytokines IL-1 and TNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure to LPS or thrombin, and by expression of certain viral gene products. As a transcription factor, NF-KB regulates the expression of genes involved in immune cell activation, control of apoptosis (NF-KB appears to shield cells from apoptosis), B and T-cell development, anti-viral and antimicrobial responses, and multiple stress responses.

In non-stimulated conditions, NF-KB is retained in the cytoplasm with I-KB (Inhibitor KB). However, upon stimulation, I-KB is phosphorylated and degraded, causing NF-KB to shuttle to the nucleus, thereby activating transcription of target genes. Target genes activated by NF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

Due to its central role and ability to respond to a range of stimuli, reporter constructs utilizing the NF-KB promoter element are used to screen the supernatants produced in Example 42. Activators or inhibitors of NF-KB would be useful in detecting, preventing, diagnosing, prognosticating, treating, and/or ameliorating diseases. For example, inhibitors of NF-KB could be used to treat those diseases related to the acute or chronic activation of NF-KB, such as rheumatoid arthritis.

To construct a vector containing the NF-KB promoter element, a PCR based strategy is employed. The upstream primer contains four tandem copies of the NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:243), 18 by of sequence complementary to the 5′ end of the SV40 early promoter sequence, and is flanked with an XhoI site: 5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATC CTGCCATCTCAATTAG:3′ (SEQ ID NO:244)

The downstream primer is complementary to the 3′ end of the SV40 promoter and is flanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO:239)

PCR amplification is performed using the SV40 promoter template present in the pB-gal:promoter plasmid obtained from CLONTECH™. The resulting PCR fragment is digested with XhoI and Hind III and subcloned into BLSK2−. (STRATAGENE™) Sequencing with the T7 and T3 primers confirms the insert contains the following sequence: 5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCTGCCA TCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTC CGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGG CCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGG CCTAGGCTTTTGCAAAAAGCTT:3′ (SEQ ID NO:245)

Next, replace the SV40 minimal promoter element present in the pSEAP2-promoter plasmid (CLONTECH™) with this NF-KB/SV40 fragment using XhoI and HindIII. However, this vector does not contain a neomycin resistance gene, and therefore, is not preferred for mammalian expression systems.

In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAP cassette is removed from the above NF-KB/SEAP vector using restriction enzymes SalI and NotI, and inserted into a vector containing neomycin resistance. Particularly, the NF-KB/SV40/SEAP cassette was inserted into pGFP-1 (CLONTECH™), replacing the GFP gene, after restricting pGFP-1 with SalI and NotI.

Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells are created and maintained according to the protocol described in Example 44. Similarly, the method for assaying supernatants with these stable Jurkat T-cells is also described in Example 44. As a positive control, exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10, and H11, with a 5-10 fold activation typically observed.

Example 48 Assay for SEAP Activity

As a reporter molecule for the assays described in Examples 44-47, SEAP activity is assayed using the Tropix Phospho-light Kit (Cat. BP-400) according to the following general procedure. The Tropix Phospho-light Kit supplies the Dilution, Assay, and Reaction Buffers used below.

Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 μl of 2.5× dilution buffer into Optiplates containing 35 μl of a supernatant. Seal the plates with a plastic sealer and incubate at 65 degree C. for 30 min. Separate the Optiplates to avoid uneven heating.

Cool the samples to room temperature for 15 minutes. Empty the dispenser and prime with the Assay Buffer. Add 50 ml Assay Buffer and incubate at room temperature 5 min. Empty the dispenser and prime with the Reaction Buffer (see the Table below). Add 50 μl Reaction Buffer and incubate at room temperature for 20 minutes. Since the intensity of the chemiluminescent signal is time dependent, and it takes about 10 minutes to read 5 plates on a luminometer, thus one should treat 5 plates at each time and start the second set 10 minutes later.

Read the relative light unit in the luminometer. Set H12 as blank, and print the results. An increase in chemiluminescence indicates reporter activity.

Reaction Buffer Formulation: # of plates Rxn buffer diluent (ml) CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 16 90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 185 9.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 215 10.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 245 12.25 48 250 12.5 49 255 12.75 50 260 13

Example 49 High-Throughput Screening Assay Identifying Changes in Small Molecule Concentration and Membrane Permeability

Binding of a ligand to a receptor is known to alter intracellular levels of small molecules, such as calcium, potassium, sodium, and pH, as well as alter membrane potential. These alterations can be measured in an assay to identify supernatants which bind to receptors of a particular cell. Although the following protocol describes an assay for calcium, this protocol can easily be modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule which is detectable by a fluorescent probe.

The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) to measure changes in fluorescent molecules (Molecular Probes) that bind small molecules. Clearly, any fluorescent molecule detecting a small molecule can be used instead of the calcium fluorescent molecule, fluo-4 (Molecular Probes, Inc.; catalog no. F-14202), used here.

For adherent cells, seed the cells at 10,000-20,000 cells/well in a Co-star black 96-well plate with clear bottom. The plate is incubated in a CO₂ incubator for 20 hours. The adherent cells are washed two times in Biotek washer with 200 μl of HBSS (Hank's Balanced Salt Solution) leaving 100 μl of buffer after the final wash.

A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. To load the cells with fluo-4, 50 μl of 12 μg/ml fluo-4 is added to each well. The plate is incubated at 37 degrees C. in a CO₂ incubator for 60 min. The plate is washed four times in the Biotek washer with HBSS leaving 100 μl of buffer.

For non-adherent cells, the cells are spun down from culture media. Cells are re-suspended to 2−5×10⁶ cells/ml with HBSS in a 50-ml conical tube. 4 μl of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is added to each ml of cell suspension. The tube is then placed in a 37 degrees C. water bath for 30-60 min. The cells are washed twice with HBSS, resuspended to 1×10⁶ cells/ml, and dispensed into a microplate, 100 μl/well. The plate is centrifuged at 1000 rpm for 5 min. The plate is then washed once in Denley Cell Wash with 200 μl, followed by an aspiration step to 100 μl final volume.

For a non-cell based assay, each well contains a fluorescent molecule, such as fluo-4. The supernatant is added to the well, and a change in fluorescence is detected.

To measure the fluorescence of intracellular calcium, the FLIPR is set for the following parameters: (1) System gain is 300-800 mW; (2) Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is 488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 μl. Increased emission at 530 nm indicates an extracellular signaling event caused by the a molecule, either polypeptide of the present invention or a molecule induced by polypeptide of the present invention, which has resulted in an increase in the intracellular Ca⁺⁺ concentration.

Example 50 High-Throughput Screening Assay Identifying Tyrosine Kinase Activity

The Protein Tyrosine Kinases (PTK) represent a diverse group of transmembrane and cytoplasmic kinases. Within the Receptor Protein Tyrosine Kinase RPTK) group are receptors for a range of mitogenic and metabolic growth factors including the PDGF, FGF, EGF, NGF, HGF and Insulin receptor subfamilies. In addition there are a large family of RPTKs for which the corresponding ligand is unknown. Ligands for RPTKs include mainly secreted small proteins, but also membrane-bound and extracellular matrix proteins.

Activation of RPTK by ligands involves ligand-mediated receptor dimerization, resulting in transphosphorylation of the receptor subunits and activation of the cytoplasmic tyrosine kinases. The cytoplasmic tyrosine kinases include receptor associated tyrosine kinases of the src-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked and cytosolic protein tyrosine kinases, such as the Jak family, members of which mediate signal transduction triggered by the cytokine superfamily of receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

Because of the wide range of known factors capable of stimulating tyrosine kinase activity, identifying whether polypeptide of the present invention or a molecule induced by polypeptide of the present invention is capable of activating tyrosine kinase signal transduction pathways is of interest. Therefore, the following protocol is designed to identify such molecules capable of activating the tyrosine kinase signal transduction pathways.

Seed target cells (e.g., primary keratinocytes) at a density of approximately 25,000 cells per well in a 96 well LOPRODYNE™ Silent Screen Plates purchased from Nalge Nunc (Naperville, Ill.). The plates are sterilized with two 30 minute rinses with 100% ethanol, rinsed with water and dried overnight. Some plates are coated for 2 hr with 100 ml of cell culture grade type I collagen (50 mg/ml), gelatin (2%) or polylysine (50 mg/ml), all of which can be purchased from SIGMA™ Chemicals (St. Louis, Mo.) or 10% MATRIGEL™ purchased from Becton Dickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at 4 degree C. Cell growth on these plates is assayed by seeding 5,000 cells/well in growth medium and indirect quantitation of cell number through use of ALAMAR BLUE™ as described by the manufacturer Alamar Biosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers #3071 from Becton Dickinson (Bedford, Mass.) are used to cover the LOPRODYNE™ Silent Screen Plates. Falcon Microtest III cell culture plates can also be used in some proliferation experiments.

To prepare extracts, A431 cells are seeded onto the nylon membranes of LOPRODYNE™ plates (20,000/200 ml/well) and cultured overnight in complete medium. Cells are quiesced by incubation in serum-free basal medium for 24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50 μl of the supernatant produced in Example 42, the medium was removed and 100 ml of extraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100, 0.1% SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and a cocktail of protease inhibitors (#1836170) obtained from Boeheringer Mannheim (Indianapolis, Ind.)) is added to each well and the plate is shaken on a rotating shaker for 5 minutes at 4° C. The plate is then placed in a vacuum transfer manifold and the extract filtered through the 0.45 mm membrane bottoms of each well using house vacuum. Extracts are collected in a 96-well catch/assay plate in the bottom of the vacuum manifold and immediately placed on ice. To obtain extracts clarified by centrifugation, the content of each well, after detergent solubilization for 5 minutes, is removed and centrifuged for 15 minutes at 4 degree C. at 16,000×g.

Test the filtered extracts for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one method is described here.

Generally, the tyrosine kinase activity of a supernatant is evaluated by determining its ability to phosphorylate a tyrosine residue on a specific substrate (a biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (corresponding to amino acids 6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a range of tyrosine kinases and are available from BOEHRINGER™ Mannheim.

The tyrosine kinase reaction is set up by adding the following components in order. First, add 10 μl of 5 uM Biotinylated Peptide, then 10 μl ATP/Mg₂₊ (5 mM ATP/50 mM MgCl₂), then 10 μl of 5× Assay Buffer (40 mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mM EGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 μl of Sodium Vanadate (1 mM), and then 5 μl of water. Mix the components gently and preincubate the reaction mix at 30 degree C. for 2 min. Initial the reaction by adding 10 μl of the control enzyme or the filtered supernatant.

The tyrosine kinase assay reaction is then terminated by adding 10 μl of 120 mm EDTA and place the reactions on ice.

Tyrosine kinase activity is determined by transferring 50 μl aliquot of reaction mixture to a microtiter plate (MTP) module and incubating at 37 degree C. for 20 min. This allows the streptavidin coated 96 well plate to associate with the biotinylated peptide. Wash the MTP module with 300 ul/well of PBS four times. Next add 75 μl of anti-phosphotyrosine antibody conjugated to horse radish peroxidase (anti-P-Tyr-POD(0.5 u/ml)) to each well and incubate at 37 degree C. for one hour. Wash the well as above.

Next add 100 μl of peroxidase substrate solution (BOEHRINGER™ Mannheim) and incubate at room temperature for at least 5 mins (up to 30 min). Measure the absorbance of the sample at 405 nm by using ELISA reader. The level of bound peroxidase activity is quantitated using an ELISA reader and reflects the level of tyrosine kinase activity.

Example 51 High-Throughput Screening Assay Identifying Phosphorylation Activity

As a potential alternative and/or complement to the assay of protein tyrosine kinase activity described in Example 50, an assay which detects activation (phosphorylation) of major intracellular signal transduction intermediates can also be used. For example, as described below one particular assay can detect tyrosine phosphorylation of the Erk-1 and Erk-2 kinases. However, phosphorylation of other molecules, such as Raf, JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specific kinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine, phosphotyrosine, or phosphothreonine molecule, can be detected by substituting these molecules for Erk-1 or Erk-2 in the following assay.

Specifically, assay plates are made by coating the wells of a 96-well ELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp, (RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBS for 1 hr at RT. The protein G plates are then treated with 2 commercial monoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT) (Santa Cruz Biotechnology). (To detect other molecules, this step can easily be modified by substituting a monoclonal antibody detecting any of the above described molecules.) After 3-5 rinses with PBS, the plates are stored at 4 degree C. until use.

A431 cells are seeded at 20,000/well in a 96-well LOPRODYNE™ filterplate and cultured overnight in growth medium. The cells are then starved for 48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50 μl of the supernatants obtained in Example 42 for 5-20 minutes. The cells are then solubilized and extracts filtered directly into the assay plate.

After incubation with the extract for 1 hr at RT, the wells are again rinsed. As a positive control, a commercial preparation of MAP kinase (10 ng/well) is used in place of A431 extract. Plates are then treated with a commercial polyclonal (rabbit) antibody (1 ug/ml) which specifically recognizes the phosphorylated epitope of the Erk-1 and Erk-2 kinases (1 hr at RT). This antibody is biotinylated by standard procedures. The bound polyclonal antibody is then quantitated by successive incubations with Europium-streptavidin and Europium fluorescence enhancing reagent in the Wallac DELFIA instrument (time-resolved fluorescence). An increased fluorescent signal over background indicates a phosphorylation by polypeptide of the present invention or a molecule induced by polypeptide of the present invention.

Example 52 Assay for the Stimulation of Bone Marrow CD34+ Cell Proliferation

This assay is based on the ability of human CD34+ to proliferate in the presence of hematopoietic growth factors and evaluates the ability of isolated polypeptides expressed in mammalian cells to stimulate proliferation of CD34+ cells.

It has been previously shown that most mature precursors will respond to only a single signal. More immature precursors require at least two signals to respond. Therefore, to test the effect of polypeptides on hematopoietic activity of a wide range of progenitor cells, the assay contains a given polypeptide in the presence or absence of other hematopoietic growth factors. Isolated cells are cultured for 5 days in the presence of Stem Cell Factor (SCF) in combination with tested sample. SCF alone has a very limited effect on the proliferation of bone marrow (BM) cells, acting in such conditions only as a “survival” factor. However, combined with any factor exhibiting stimulatory effect on these cells (e.g., IL-3), SCF will cause a synergistic effect. Therefore, if the tested polypeptide has a stimulatory effect on hematopoietic progenitors, such activity can be easily detected. Since normal BM cells have a low level of cycling cells, it is likely that any inhibitory effect of a given polypeptide, or agonists or antagonists thereof, might not be detected. Accordingly, assays for an inhibitory effect on progenitors is preferably tested in cells that are first subjected to in vitro stimulation with SCF+IL+3, and then contacted with the compound that is being evaluated for inhibition of such induced proliferation.

Briefly, CD34+ cells are isolated using methods known in the art. The cells are thawed and resuspended in medium (QBSF 60 serum-free medium with 1% L-glutamine (500 ml) Quality Biological, Inc., Gaithersburg, Md. Cat#160-204-101). After several gentle centrifugation steps at 200×g, cells are allowed to rest for one hour. The cell count is adjusted to 2.5×10⁵ cells/ml. During this time, 100 μl of sterile water is added to the peripheral wells of a 96-well plate. The cytokines that can be tested with a given polypeptide in this assay is rhSCF (R&D Systems, Minneapolis, Minn., Cat#255-SC) at 50 ng/ml alone and in combination with rhSCF and rhIL-3 (R&D Systems, Minneapolis, Minn., Cat#203-ML) at 30 ng/ml. After one hour, 10 μl of prepared cytokines, 50 μl of the supernatants prepared in Example 42 (supernatants at 1:2 dilution=50 μl) and 20 μl of diluted cells are added to the media which is already present in the wells to allow for a final total volume of 100 μl. The plates are then placed in a 37° C./5% CO₂ incubator for five days.

Eighteen hours before the assay is harvested, 0.5 μCi/well of [3H] Thymidine is added in a 10 μl volume to each well to determine the proliferation rate. The experiment is terminated by harvesting the cells from each 96-well plate to a filtermat using the Tomtec Harvester 96. After harvesting, the filtermats are dried, trimmed and placed into OMNIFILTER™ assemblies consisting of one OMNIFILTER™ plate and one OMNIFILTER™ Tray. 60 μl MICROSCINT™ is added to each well and the plate sealed with TopSeal-A press-on sealing film. A bar code 15 sticker is affixed to the first plate for counting. The sealed plates are then loaded and the level of radioactivity determined via the Packard Top Count and the printed data collected for analysis. The level of radioactivity reflects the amount of cell proliferation.

The studies described in this example test the activity of a given polypeptide to stimulate bone marrow CD34+ cell proliferation. One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof. As a nonlimiting example, potential antagonists tested in this assay would be expected to inhibit cell proliferation in the presence of cytokines and/or to increase the inhibition of cell proliferation in the presence of cytokines and a given polypeptide. In contrast, potential agonists tested in this assay would be expected to enhance cell proliferation and/or to decrease the inhibition of cell proliferation in the presence of cytokines and a given polypeptide.

The ability of a gene to stimulate the proliferation of bone marrow CD34+ cells indicates that polynucleotides and polypeptides corresponding to the gene are useful for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections above, and elsewhere herein.

Example 53 Assay for Extracellular Matrix Enhanced Cell Response (EMECR)

The objective of the Extracellular Matrix Enhanced Cell Response (EMECR) assay is to identify gene products (e.g., isolated polypeptides) that act on the hematopoietic stem cells in the context of the extracellular matrix (ECM) induced signal.

Cells respond to the regulatory factors in the context of signal(s) received from the surrounding microenvironment. For example, fibroblasts, and endothelial and epithelial stem cells fail to replicate in the absence of signals from the ECM. Hematopoietic stem cells can undergo self-renewal in the bone marrow, but not in in vitro suspension culture. The ability of stem cells to undergo self-renewal in vitro is dependent upon their interaction with the stromal cells and the ECM protein fibronectin (fn). Adhesion of cells to fn is mediated by the α₅.β₁ and α₄.β₁ integrin receptors, which are expressed by human and mouse hematopoietic stem cells. The factor(s) which integrate with the ECM environment and are responsible for stimulating stem cell self-renewal have not yet been identified. Discovery of such factors should be of great interest in gene therapy and bone marrow transplant applications

Briefly, polystyrene, non tissue culture treated, 96-well plates are coated with fn fragment at a coating concentration of 0.2 μg/cm². Mouse bone marrow cells are plated (1,000 cells/well) in 0.2 ml of serum-free medium. Cells cultured in the presence of IL-3 (5 ng/ml)+SCF (50 ng/ml) would serve as the positive control, conditions under which little self-renewal but pronounced differentiation of the stem cells is to be expected. Gene products of the invention (e.g., including, but not limited to, polynucleotides and polypeptides of the present invention, and supernatants produced in Example 42), are tested with appropriate negative controls in the presence and absence of SCF (5.0 ng/ml), where test factor supernatants represent 10% of the total assay volume. The plated cells are then allowed to grow by incubating in a low oxygen environment (5% CO₂, 7% O₂, and 88% N₂) tissue culture incubator for 7 days. The number of proliferating cells within the wells is then quantitated by measuring thymidine incorporation into cellular DNA. Verification of the positive hits in the assay will require phenotypic characterization of the cells, which can be accomplished by scaling up of the culture system and using appropriate antibody reagents against cell surface antigens and FACScan.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.

If a particular polypeptide of the present invention is found to be a stimulator of hematopoietic progenitors, polynucleotides and polypeptides corresponding to the gene encoding said polypeptide may be useful for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of disorders affecting the immune system and hematopoiesis. Representative uses are described in the “Immune Activity” and “Infectious Disease” sections above, and elsewhere herein. The gene product may also be useful in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.

Additionally, the polynucleotides and/or polypeptides of the gene of interest and/or agonists and/or antagonists thereof, may also be employed to inhibit the proliferation and differentiation of hematopoietic cells and therefore may be employed to protect bone marrow stem cells from chemotherapeutic agents during chemotherapy. This antiproliferative effect may allow administration of higher doses of chemotherapeutic agents and, therefore, more effective chemotherapeutic treatment.

Moreover, polynucleotides and polypeptides corresponding to the gene of interest may also be useful for the detection, prevention, diagnosis, prognostication, treatment, and/or amelioration of hematopoietic related disorders such as, for example, anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia since stromal cells are important in the production of cells of hematopoietic lineages. The uses include bone marrow cell ex-vivo culture, bone marrow transplantation, bone marrow reconstitution, radiotherapy or chemotherapy of neoplasia.

Example 54 Human Dermal Fibroblast and Aortic Smooth Muscle Cell Proliferation

The polypeptide of interest is added to cultures of normal human dermal fibroblasts (NHDF) and human aortic smooth muscle cells (AoSMC) and two co-assays are performed with each sample. The first assay examines the effect of the polypeptide of interest on the proliferation of normal human dermal fibroblasts (NHDF) or aortic smooth muscle cells (AoSMC). Aberrant growth of fibroblasts or smooth muscle cells is a part of several pathological processes, including fibrosis, and restenosis. The second assay examines IL6 production by both NHDF and SMC. IL6 production is an indication of functional activation. Activated cells will have increased production of a number of cytokines and other factors, which can result in a proinflammatory or immunomodulatory outcome. Assays are run with and without co-TNFa stimulation, in order to check for costimulatory or inhibitory activity.

Briefly, on day 1, 96-well black plates are set up with 1000 cells/well (NHDF) or 2000 cells/well (AoSMC) in 100 μl culture media. NHDF culture media contains: Clonetics FB basal media, 1 mg/ml hFGF, 5 mg/ml insulin, 50 mg/ml gentamycin, 2% FBS, while AoSMC culture media contains Clonetics SM basal media, 0.5 μg/ml hEGF, 5 mg/ml insulin, 1 μg/ml hFGF, 50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 5% FBS. After incubation at 37° C. for at least 4-5 hours culture media is aspirated and replaced with growth arrest media. Growth arrest media for NHDF contains fibroblast basal media, 50 mg/ml gentamycin, 2% FBS, while growth arrest media for AoSMC contains SM basal media, 50 mg/ml gentamycin, 50 μg/ml Amphotericin B, 0.4% FBS. Incubate at 37° C. until day 2.

On day 2, serial dilutions and templates of the polypeptide of interest are designed such that they always include media controls and known-protein controls. For both stimulation and inhibition experiments, proteins are diluted in growth arrest media. For inhibition experiments, TNFa is added to a final concentration of 2 ng/ml (NHDF) or 5 ng/ml (AoSMC). Add ⅓ vol media containing controls or polypeptides of the present invention and incubate at 37° C./5% CO₂ until day 5.

Transfer 60 μl from each well to another labeled 96-well plate, cover with a plate-sealer, and store at 4° C. until Day 6 (for IL6 ELISA). To the remaining 100 μl in the cell culture plate, aseptically add ALAMAR BLUE™ in an amount equal to 10% of the culture volume (10 μl). Return plates to incubator for 3 to 4 hours. Then measure fluorescence with excitation at 530 nm and emission at 590 nm using the CYTOFLUOR™. This yields the growth stimulation/inhibition data.

On day 5, the IL6 ELISA is performed by coating a 96 well plate with 50-100 μl/well of Anti-Human IL6 Monoclonal antibody diluted in PBS, pH 7.4, incubate ON at room temperature.

On day 6, empty the plates into the sink and blot on paper towels. Prepare Assay Buffer containing PBS with 4% BSA. Block the plates with 200 μl/well of Pierce Super Block blocking buffer in PBS for 1-2 hr and then wash plates with wash buffer (PBS, 0.05% Tween-20). Blot plates on paper towels. Then add 50 μl/well of diluted Anti-Human IL-6 Monoclonal, Biotin-labeled antibody at 0.50 mg/ml. Make dilutions of IL-6 stock in media (30, 10, 3, 1, 0.3, 0 ng/ml). Add duplicate samples to top row of plate. Cover the plates and incubate for 2 hours at RT on shaker.

Plates are washed with wash buffer and blotted on paper towels. Dilute EU-labeled Streptavidin 1:1000 in Assay buffer, and add 100 μl/well. Cover the plate and incubate 1 h at RT. Plates are again washed with wash buffer and blotted on paper towels.

Add 100 μl/well of Enhancement Solution. Shake for 5 minutes. Read the plate on the Wallac DELFIA Fluorometer. Readings from triplicate samples in each assay were tabulated and averaged.

A positive result in this assay suggests AoSMC cell proliferation and that the polypeptide of the present invention may be involved in dermal fibroblast proliferation and/or smooth muscle cell proliferation. A positive result also suggests many potential uses of polypeptides, polynucleotides, agonists and/or antagonists of the polynucleotide/polypeptide of the present invention which gives a positive result. For example, inflammation and immune responses, wound healing, and angiogenesis, as detailed throughout this specification. Particularly, polypeptides of the present invention and polynucleotides of the present invention may be used in wound healing and dermal regeneration, as well as the promotion of vasculogenesis, both of the blood vessels and lymphatics. The growth of vessels can be used in the treatment of, for example, cardiovascular diseases. Additionally, antagonists of polypeptides and polynucleotides of the invention may be useful in treating diseases, disorders, and/or conditions which involve angiogenesis by acting as an anti-vascular agent (e.g., anti-angiogenesis). These diseases, disorders, and/or conditions are known in the art and/or are described herein, such as, for example, malignancies, solid tumors, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion fractures; scleroderma; trachoma; vascular adhesions; myocardial angiogenesis; coronary collaterals; cerebral collaterals; arteriovenous malformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; fibromuscular dysplasia; wound granulation; Crohn's disease; and atherosclerosis. Moreover, antagonists of polypeptides and polynucleotides of the invention may be useful in treating anti-hyperproliferative diseases and/or anti-inflammatory known in the art and/or described herein.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.

Example 55 Cellular Adhesion Molecule (CAM) Expression on Endothelial Cells

The recruitment of lymphocytes to areas of inflammation and angiogenesis involves specific receptor-ligand interactions between cell surface adhesion molecules (CAMs) on lymphocytes and the vascular endothelium. The adhesion process, in both normal and pathological settings, follows a multi-step cascade that involves intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) expression on endothelial cells (EC). The expression of these molecules and others on the vascular endothelium determines the efficiency with which leukocytes may adhere to the local vasculature and extravasate into the local tissue during the development of an inflammatory response. The local concentration of cytokines and growth factor participate in the modulation of the expression of these CAMs.

Briefly, endothelial cells (e.g., Human Umbilical Vein Endothelial cells (HUVECs)) are grown in a standard 96 well plate to confluence, growth medium is removed from the cells and replaced with 100 μl of 199 Medium (10% fetal bovine serum (FBS)). Samples for testing and positive or negative controls are added to the plate in triplicate (in 10 μl volumes). Plates are then incubated at 37° C. for either 5 h (selectin and integrin expression) or 24 h (integrin expression only). Plates are aspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS (with Ca++ and Mg++) is added to each well. Plates are held at 4° C. for 30 min. Fixative is removed from the wells and wells are washed 1× with PBS(+Ca,Mg)+0.5% BSA and drained. 10 μl of diluted primary antibody is added to the test and control wells. Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin are used at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stock antibody). Cells are incubated at 37° C. for 30 min. in a humidified environment. Wells are washed three times with PBS (+Ca,Mg)+0.5% BSA. 20 μl of diluted Extravidin®-Alkaline Phosphatase (1:5,000 dilution, referred to herein as the working dilution) are added to each well and incubated at 37° C. for 30 min. Wells are washed three times with PBS (+Ca, Mg)+0.5% BSA. Dissolve 1 tablet of p-Nitrophenol Phosphate pNPP per 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Standard wells in triplicate are prepared from the working dilution of the ExtrAvidin®-Alkaline Phosphotase in glycine buffer: 1:5,000 (10⁰)>10^(−0.5)>10⁻¹>10^(−1.5)0.5 μl of each dilution is added to triplicate wells and the resulting AP content in each well is 5.50 ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent is then added to each of the standard wells. The plate is incubated at 37° C. for 4 h. A volume of 50 μl of 3M NaOH is added to all wells. The plate is read on a plate reader at 405 nm using the background subtraction option on blank wells filled with glycine buffer only. Additionally, the template is set up to indicate the concentration of AP-conjugate in each standard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are indicated as amount of bound AP-conjugate in each sample.

Example 56 ALAMAR BLUE™ Endothelial Cells Proliferation Assay

This assay may be used to quantitatively determine protein mediated inhibition of bFGF-induced proliferation of Bovine Lymphatic Endothelial Cells (LECs), Bovine Aortic Endothelial Cells (BAECs) or Human Microvascular Uterine Myometrial Cells (UTMECs). This assay incorporates a fluorometric growth indicator based on detection of metabolic activity. A standard ALAMAR BLUE™ Proliferation Assay is prepared in EGM-2MV with 10 ng/ml of bFGF added as a source of endothelial cell stimulation. This assay may be used with a variety of endothelial cells with slight changes in growth medium and cell concentration. Dilutions of the protein batches to be tested are diluted as appropriate. Serum-free medium (GIBCO SFM) without bFGF is used as a non-stimulated control and Angiostatin or TSP-1 are included as a known inhibitory controls.

Briefly, LEC, BAECs or UTMECs are seeded in growth media at a density of 5000 to 2000 cells/well in a 96 well plate and placed at 37° C. overnight. After the overnight incubation of the cells, the growth media is removed and replaced with GIBCO EC-SFM. The cells are treated with the appropriate dilutions of the protein of interest or control protein sample(s) (prepared in SFM) in triplicate wells with additional bFGF to a concentration of 10 ng/ml. Once the cells have been treated with the samples, the plate(s) is/are placed back in the 37° C. incubator for three days. After three days 10 ml of stock ALAMAR BLUE™ (Biosource Cat#DAL1100) is added to each well and the plate(s) is/are placed back in the 37° C. incubator for four hours. The plate(s) are then read at 530 nm excitation and 590 nm emission using the CYTOFLUOR™ fluorescence reader. Direct output is recorded in relative fluorescence units.

ALAMAR BLUE™ is an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. As cells grow in culture, innate metabolic activity results in a chemical reduction of the immediate surrounding environment. Reduction related to growth causes the indicator to change from oxidized (non-fluorescent blue) form to reduced (fluorescent red) form (i.e., stimulated proliferation will produce a stronger signal and inhibited proliferation will produce a weaker signal and the total signal is proportional to the total number of cells as well as their metabolic activity). The background level of activity is observed with the starvation medium alone. This is compared to the output observed from the positive control samples (bFGF in growth medium) and protein dilutions.

Example 57 Detection of Inhibition of a Mixed Lymphocyte Reaction

This assay can be used to detect and evaluate inhibition of a Mixed Lymphocyte Reaction (MLR) by gene products (e.g., isolated polypeptides). Inhibition of a MLR may be due to a direct effect on cell proliferation and viability, modulation of costimulatory molecules on interacting cells, modulation of adhesiveness between lymphocytes and accessory cells, or modulation of cytokine production by accessory cells. Multiple cells may be targeted by these polypeptides since the peripheral blood mononuclear fraction used in this assay includes T, B and natural killer lymphocytes, as well as monocytes and dendritic cells.

Polypeptides of interest found to inhibit the MLR may find application in diseases associated with lymphocyte and monocyte activation or proliferation. These include, but are not limited to, diseases such as asthma, arthritis, diabetes, inflammatory skin conditions, psoriasis, eczema, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, inflammatory bowel disease, crohn's disease, ulcerative colitis, arteriosclerosis, cirrhosis, graft vs. host disease, host vs. graft disease, hepatitis, leukemia and lymphoma.

Briefly, PBMCs from human donors are purified by density gradient centrifugation using Lymphocyte Separation Medium (LSM®, density 1.0770 g/ml, Organon Teknika Corporation, West Chester, Pa.). PBMCs from two donors are adjusted to 2×10⁶ cells/ml in RPMI-1640 (LIFE TECHNOLOGIES™, Grand Island, N.Y.) supplemented with 10% FCS and 2 mM glutamine. PBMCs from a third donor are adjusted to 2×10⁵ cells/ml. Fifty microliters of PBMCs from each donor is added to wells of a 96-well round bottom microtiter plate. Dilutions of test materials (50 μl) are added in triplicate to microtiter wells. Test samples (of the protein of interest) are added for final dilution of 1:4; rhuIL-2 (R&D Systems, Minneapolis, Minn., catalog number 202-IL) is added to a final concentration of 1 μg/ml; anti-CD4 mAb (R&D Systems, clone 34930.11, catalog number MAB379) is added to a final concentration of 10 μg/ml. Cells are cultured for 7-8 days at 37° C. in 5% CO₂, and 1 μC. of [³H] thymidine is added to wells for the last 16 hrs of culture. Cells are harvested and thymidine incorporation determined using a Packard TopCount. Data is expressed as the mean and standard deviation of triplicate determinations.

Samples of the protein of interest are screened in separate experiments and compared to the negative control treatment, anti-CD4 mAb, which inhibits proliferation of lymphocytes and the positive control treatment, IL-2 (either as recombinant material or supernatant), which enhances proliferation of lymphocytes.

One skilled in the art could easily modify the exemplified studies to test the activity of polynucleotides (e.g., gene therapy), antibodies, agonists, and/or antagonists and fragments and variants thereof.

Example 58 Assays for Protease Activity

The following assay may be used to assess protease activity of the polypeptides of the invention.

Gelatin and casein zymography are performed essentially as described (Heusen et al., Anal. Biochem., 102:196-202 (1980); Wilson et al., Journal of Urology, 149:653-658 (1993)). Samples are run on 10% polyacryamide/0.1% SDS gels containing 1% gelain orcasein, soaked in 2.5% triton at room temperature for 1 hour, and in 0.1M glycine, pH 8.3 at 37° C. 5 to 16 hours. After staining in amido black areas of proteolysis appear as clear areas against the blue-black background. Trypsin (SIGMA™ T8642) is used as a positive control.

Protease activity is also determined by monitoring the cleavage of n-a-benzoyl-L-arginine ethyl ester (BAEE) (SIGMA™ B-4500). Reactions are set up in (25 mMNaPO₄,1 mM EDTA, and 1 mM BAEE), pH 7.5. Samples are added and the change in absorbance at 260 nm is monitored on the Beckman DU-6 spectrophotometer in the time-drive mode. Trypsin is used as a positive control.

Additional assays based upon the release of acid-soluble peptides from casein or hemoglobin measured as absorbance at 280 nm or colorimetrically using the Folin method are performed as described in Bergmeyer, et al., Methods of Enzymatic Analysis, 5 (1984). Other assays involve the solubilization of chromogenic substrates (Ward, Applied Science, 251-317 (1983)).

Example 59 Identifying Serine Protease Substrate Specificity

Methods known in the art or described herein may be used to determine the substrate specificity of the polypeptides of the present invention having serine protease activity. A preferred method of determining substrate specificity is by the use of positional scanning synthetic combinatorial libraries as described in GB 2 324 529 (incorporated herein in its entirety).

Example 60 Ligand Binding Assays

The following assay may be used to assess ligand binding activity of the polypeptides of the invention.

Ligand binding assays provide a direct method for ascertaining receptor pharmacology and are adaptable to a high throughput format. The purified ligand for a polypeptide is radiolabeled to high specific activity (50-2000 Ci/mmol) for binding studies. A determination is then made that the process of radiolabeling does not diminish the activity of the ligand towards its polypeptide. Assay conditions for buffers, ions, pH and other modulators such as nucleotides are optimized to establish a workable signal to noise ratio for both membrane and whole cell polypeptide sources. For these assays, specific polypeptide binding is defined as total associated radioactivity minus the radioactivity measured in the presence of an excess of unlabeled competing ligand. Where possible, more than one competing ligand is used to define residual nonspecific binding.

Example 61 Functional Assay in Xenopus Oocytes

Capped RNA transcripts from linearized plasmid templates encoding the polypeptides of the invention are synthesized in vitro with RNA polymerases in accordance with standard procedures. In vitro transcripts are suspended in water at a final concentration of 0.2 mg/ml. Ovarian lobes are removed from adult female toads, Stage V defolliculated oocytes are obtained, and RNA transcripts (10 ng/oocytc) are injected in a 50 nl bolus using a microinjection apparatus. Two electrode voltage clamps are used to measure the currents from individual Xenopus oocytes in response polypeptides and polypeptide agonist exposure. Recordings are made in Ca2+ free Barth's medium at room temperature. The Xenopus system can be used to screen known ligands and tissue/cell extracts for activating ligands.

Example 62 Microphysiometric Assays

Activation of a wide variety of secondary messenger systems results in extrusion of small amounts of acid from a cell. The acid formed is largely as a result of the increased metabolic activity required to fuel the intracellular signaling process. The pH changes in the media surrounding the cell are very small but are detectable by the CYTOSENSOR microphysiometer (Molecular Devices Ltd., Menlo Park, Calif.). The CYTOSENSOR is thus capable of detecting the activation of polypeptide that is coupled to an energy utilizing intracellular signaling pathway.

Example 63 Extract/Cell Supernatant Screening

A large number of mammalian receptors exist for which there remains, as yet, no cognate activating ligand (agonist). Thus, active ligands for these receptors may not be included within the ligands banks as identified to date. Accordingly, the polypeptides of the invention can also be functionally screened (using calcium, cAMP, microphysiometer, oocyte electrophysiology, etc., functional screens) against tissue extracts to identify its natural ligands. Extracts that produce positive functional responses can be sequentially subfractionated until an activating ligand is isolated and identified.

Example 64 Calcium and cAMP Functional Assays

Seven transmembrane receptors which are expressed in HEK 293 cells have been shown to be coupled functionally to activation of PLC and calcium mobilization and/or cAMP stimulation or inhibition. Basal calcium levels in the HEK 293 cells in receptor-transfected or vector control cells were observed to be in the normal, 100 nM to 200 nM, range. HEK 293 cells expressing recombinant receptors are loaded with fura 2 and in a single day>150 selected ligands or tissue/cell extracts are evaluated for agonist induced calcium mobilization. Similarly, HEK 293 cells expressing recombinant receptors are evaluated for the stimulation or inhibition of cAMP production using standard cAMP quantitation assays. Agonists presenting a calcium transient or cAMP fluctuation are tested in vector control cells to determine if the response is unique to the transfected cells expressing receptor.

Example 65 ATP-Binding Assay

The following assay may be used to assess ATP-binding activity of polypeptides of the invention.

ATP-binding activity of the polypeptides of the invention may be detected using the ATP-binding assay described in U.S. Pat. No. 5,858,719, which is herein incorporated by reference in its entirety. Briefly, ATP-binding to polypeptides of the invention is measured via photoaffinity labeling with 8-azido-ATP in a competition assay. Reaction mixtures containing 1 mg/ml of the ABC transport protein of the present invention are incubated with varying concentrations of ATP, or the non-hydrolyzable ATP analog adenyl-5′-imidodiphosphate for 10 minutes at 4° C. A mixture of 8-azido-ATP (SIGMA™ Chem. Corp., St. Louis, Mo.) plus 8-azido-ATP (³²P-ATP) (5 mCi/μmol, ICN, Irvine Calif.) is added to a final concentration of 100 μM and 0.5 ml aliquots are placed in the wells of a porcelain spot plate on ice. The plate is irradiated using a short wave 254 nm UV lamp at a distance of 2.5 cm from the plate for two one-minute intervals with a one-minute cooling interval in between. The reaction is stopped by addition of dithiothreitol to a final concentration of 2 mM. The incubations are subjected to SDS-PAGE electrophoresis, dried, and autoradiographed. Protein bands corresponding to the particular polypeptides of the invention are excised, and the radioactivity quantified. A decrease in radioactivity with increasing ATP or adenly-5′-imidodiphosphate provides a measure of ATP affinity to the polypeptides.

Example 66 Small Molecule Screening

This invention is particularly useful for screening therapeutic compounds by using the polypeptides of the invention, or binding fragments thereof, in any of a variety of drug screening techniques. The polypeptide or fragment employed in such a test may be affixed to a solid support, expressed on a cell surface, free in solution, or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. One may measure, for example, the formulation of complexes between the agent being tested and polypeptide of the invention.

Thus, the present invention provides methods of screening for drugs or any other agents which affect activities mediated by the polypeptides of the invention. These methods comprise contacting such an agent with a polypeptide of the invention or fragment thereof and assaying for the presence of a complex between the agent and the polypeptide or fragment thereof, by methods well known in the art. In such a competitive binding assay, the agents to screen are typically labeled. Following incubation, free agent is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular agent to bind to the polypeptides of the invention.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the polypeptides of the invention, and is described in great detail in European Patent Application 84/03564, published on Sep. 13, 1984, which is herein incorporated by reference in its entirety. Briefly stated, large numbers of different small molecule test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with polypeptides of the invention and washed. Bound polypeptides are then detected by methods well known in the art. Purified polypeptides are coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies may be used to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptides of the invention specifically compete with a test compound for binding to the polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide that shares one or more antigenic epitopes with a polypeptide of the invention.

Example 67 Phosphorylation Assay

In order to assay for phosphorylation activity of the polypeptides of the invention, a phosphorylation assay as described in U.S. Pat. No. 5,958,405 (which is herein incorporated by reference) is utilized. Briefly, phosphorylation activity may be measured by phosphorylation of a protein substrate using gamma-labeled ³²P-ATP and quantitation of the incorporated radioactivity using a gamma radioisotope counter. The polypeptides of the invention are incubated with the protein substrate, ³²P-ATP, and a kinase buffer. The ³²P incorporated into the substrate is then separated from free ³²P-ATP by electrophoresis, and the incorporated ³²P is counted and compared to a negative control. Radioactivity counts above the negative control are indicative of phosphorylation activity of the polypeptides of the invention.

Example 68 Detection of Phosphorylation Activity (Activation) of the Polypeptides of the Invention in the Presence of Polypeptide Ligands

Methods known in the art or described herein may be used to determine the phosphorylation activity of the polypeptides of the invention. A preferred method of determining phosphorylation activity is by the use of the tyrosine phosphorylation assay as described in U.S. Pat. No. 5,817,471 (incorporated herein by reference).

Example 69 Identification of Signal Transduction Proteins that Interact with Polypeptides of The Present Invention

The purified polypeptides of the invention are research tools for the identification, characterization and purification of additional signal transduction pathway proteins or receptor proteins. Briefly, labeled polypeptides of the invention are useful as reagents for the purification of molecules with which it interacts. In one embodiment of affinity purification, polypeptides of the invention are covalently coupled to a chromatography column. Cell-free extract derived from putative target cells, such as carcinoma tissues, is passed over the column, and molecules with appropriate affinity bind to the polypeptides of the invention. The protein complex is recovered from the column, dissociated, and the recovered molecule subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.

Example 70 IL-6 Bioassay

To test the proliferative effects of the polypeptides of the invention, the IL-6 Bioassay as described by Marz et al. is utilized (Proc. Natl. Acad. Sci., U.S.A., 95:3251-56 (1998), which is herein incorporated by reference). Briefly, IL-6 dependent B9 murine cells are washed three times in IL-6 free medium and plated at a concentration of 5,000 cells per well in 50 μA, and 50 μA of the IL-6-like polypeptide is added. After 68 hrs. at 37° C., the number of viable cells is measured by adding the tetrazolium salt thiazolyl blue (MTT) and incubating for a further 4 hrs. at 37° C. B9 cells are lysed by SDS and optical density is measured at 570 nm. Controls containing IL-6 (positive) and no cytokine (negative) are utilized. Enhanced proliferation in the test sample(s) relative to the negative control is indicative of proliferative effects mediated by polypeptides of the invention.

Example 71 Support of Chicken Embryo Neuron Survival

To test whether sympathetic neuronal cell viability is supported by polypeptides of the invention, the chicken embryo neuronal survival assay of Senaldi et at is utilized (Proc. Natl. Acad. Sci., U.S.A., 96:11458-63 (1998), which is herein incorporated by reference). Briefly, motor and sympathetic neurons are isolated from chicken embryos, resuspended in L15 medium (with 10% FCS, glucose, sodium selenite, progesterone, conalbumin, putrescine, and insulin; LIFE TECHNOLOGIES™, Rockville, Md.) and Dulbecco's modified Eagles medium [with 10% FCS, glutamine, penicillin, and 25 mM Hepes buffer (pH 7.2); LIFE TECHNOLOGIES™, Rockville, Md.], respectively, and incubated at 37° C. in 5% CO₂ in the presence of different concentrations of the purified IL-6-like polypeptide, as well as a negative control lacking any cytokine. After 3 days, neuron survival is determined by evaluation of cellular morphology, and through the use of the colorimetric assay of Mosmann (Mosmann, T., J. Immunol. Methods, 65:55-63 (1983)). Enhanced neuronal cell viability as compared to the controls lacking cytokine is indicative of the ability of the inventive purified IL-6-like polypeptide(s) to enhance the survival of neuronal cells.

Example 72 Assay for Phosphatase Activity

The following assay may be used to assess serine/threonine phosphatase (PTPase) activity of the polypeptides of the invention.

In order to assay for serine/threonine phosphatase (PTPase) activity, assays can be utilized which are widely known to those skilled in the art. For example, the serine/threonine phosphatase (PSPase) activity is measured using a PSPase assay kit from New England Biolabs, Inc. Myelin basic protein (MyBP), a substrate for PSPase, is phosphorylated on serine and threonine residues with cAMP-dependent Protein Kinase in the presence of [³²P]ATP. Protein serine/threonine phosphatase activity is then determined by measuring the release of inorganic phosphate from ³²P-labeled MyBP.

Example 73 Interaction of Serine/Threonine Phosphatases with Other Proteins

The polypeptides of the invention with serine/threonine phosphatase activity as determined in Example 72 are research tools for the identification, characterization and purification of additional interacting proteins or receptor proteins, or other signal transduction pathway proteins. Briefly, labeled polypeptide(s) of the invention is useful as a reagent for the purification of molecules with which it interacts. In one embodiment of affinity purification, polypeptide of the invention is covalently coupled to a chromatography column. Cell-free extract derived from putative target cells, such as neural or liver cells, is passed over the column, and molecules with appropriate affinity bind to the polypeptides of the invention. The polypeptides of the invention—complex is recovered from the column, dissociated, and the recovered molecule subjected to N-terminal protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.

Example 74 Assaying for Heparanase Activity

In order to assay for heparanase activity of the polypeptides of the invention, the heparanase assay described by Vlodaysky et al is utilized (Vlodaysky, I., et al., Nat. Med., 5:793-802 (1999)). Briefly, cell lysates, conditioned media or intact cells (1×10⁶ cells per 35-mm dish) are incubated for 18 hrs at 37° C., pH 6.2-6.6, with ³⁵S-labeled ECM or soluble ECM derived peak I proteoglycans. The incubation medium is centrifuged and the supernatant is analyzed by gel filtration on a Sepharose CL-6B column (0.9×30 cm). Fractions are eluted with PBS and their radioactivity is measured. Degradation fragments of heparan sulfate side chains are eluted from Sepharose 6B at 0.5<K_(av)<0.8 (peak II). Each experiment is done at least three times. Degradation fragments corresponding to “peak II,” as described by Vlodaysky et al., is indicative of the activity of the polypeptides of the invention in cleaving heparan sulfate.

Example 75 Immobilization of Biomolecules

This example provides a method for the stabilization of polypeptides of the invention in non-host cell lipid bilayer constucts (see, e.g., Bieri et al., Nature Biotech 17:1105-1108 (1999), hereby incorporated by reference in its entirety herein) that can be adapted for the study of polypeptides of the invention in the various functional assays described above. Briefly, carbohydrate-specific chemistry for biotinylation is used to confine a biotin tag to the extracellular domain of the polypeptides of the invention, thus allowing uniform orientation upon immobilization. A 50 uM solution of polypeptides of the invention in washed membranes is incubated with 20 mM NaIO4 and 1.5 mg/ml (4 mM) BACH or 2 mg/ml (7.5 mM) biotin-hydrazide for 1 hr at room temperature (reaction volume, 150 ul). Then the sample is dialyzed (Pierce Slidealizer Cassett, 10 kDa cutoff; Pierce Chemical Co., Rockford Ill.) at 4° C. first for 5 h, exchanging the buffer after each hour, and finally for 12 h against 500 ml buffer R (0.15 M NaCl, 1 mM MgCl₂, 10 mM sodium phosphate, pH7). Just before addition into a cuvette, the sample is diluted 1:5 in buffer ROG50 (Buffer R supplemented with 50 mM octylglucoside).

Example 76 TAQMAN®

Quantitative PCR (QPCR). Total RNA from cells in culture are extracted by TRIZOL™ separation as recommended by the supplier (LifeTechnologies). (Total RNA is treated with DNase I (LIFE TECHNOLOGIES™) to remove any contaminating genomic DNA before reverse transcription.) Total RNA (50 ng) is used in a one-step, 50 ul, RT-QPCR, consisting of TaqMan® Buffer A (Perkin-Elmer; 50 mM KCl/10 mM Tris, pH 8.3), 5.5 mM MgCl₂, 240 μM each dNTP, 0.4 units RNase inhibitor (PROMEGA™), 8% glycerol, 0.012% Tween-20, 0.05% gelatin, 0.3 μM primers, 0.1 μM probe, 0.025units Amplitaq Gold® (Perkin-Elmer) and 2.5 units Superscript II reverse transcriptase (LIFE TECHNOLOGIES™). As a control for genomic contamination, parallel reactions are setup without reverse transcriptase. The relative abundance of (unknown) and 18S RNAs are assessed by using the Applied Biosystems Prism 7700 Sequence Detection System (Livak, K. J., Flood, S. J., Marmaro, J., Giusti, W. & Deetz, K. (1995) PCR Methods Appl. 4, 357-362). Reactions are carried out at 48° C. for 30 min, 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 s, 60° C. for 1 min. Reactions are performed in triplicate.

Primers (f & r) and FRET probes sets are designed using Primer Express Software (Perkin-Elmer). Probes are labeled at the 5′-end with the reporter dye 6-FAM and on the 3′-end with the quencher dye TAMRA (Biosource International, Camarillo, Calif. or Perkin-Elmer).

Example 77 Assays for Metalloproteinase Activity

Metalloproteinases (EC 3.4.24.−) are peptide hydrolases which use metal ions, such as Zn²⁺, as the catalytic mechanism. Metalloproteinase activity of polypeptides of the present invention can be assayed according to the following methods.

Proteolysis of Alpha-2-Macroglobulin

To confirm protease activity, purified polypeptides of the invention are mixed with the substrate alpha-2-macroglobulin (0.2 unit/ml; BOEHRINGER™ Mannheim, Germany) in 1× assay buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl₂, 25 μM ZnCl₂ and 0.05% Brij-35) and incubated at 37° C. for 1-5 days. Trypsin is used as positive control. Negative controls contain only alpha-2-macroglobulin in assay buffer. The samples are collected and boiled in SDS-PAGE sample buffer containing 5% 2-mercaptoethanol for 5-min, then loaded onto 8% SDS-polyacrylamide gel. After electrophoresis the proteins are visualized by silver staining. Proteolysis is evident by the appearance of lower molecular weight bands as compared to the negative control.

Inhibition of Alpha-2-Macroglobulin Proteolysis by Inhibitors of Metalloproteinases

Known metalloproteinase inhibitors (metal chelators (EDTA, EGTA, AND HgCl₂), peptide metalloproteinase inhibitors (TIMP-1 and TIMP-2), and commercial small molecule MMP inhibitors) are used to characterize the proteolytic activity of polypeptides of the invention. The three synthetic MMP inhibitors used are: MMP inhibitor I, [IC₅₀=1.0 μM against MMP-1 and MMP-8; IC₅₀=30 μM against MMP-9; IC₅₀=150 μM against MMP-3]; MMP-3 (stromelysin-1) inhibitor I [IC₅₀=5 μM against MMP-3], and MMP-3 inhibitor II [K_(i)=130 nM against MMP-3]; inhibitors available through Calbiochem, catalog #444250, 444218, and 444225, respectively). Briefly, different concentrations of the small molecule MMP inhibitors are mixed with purified polypeptides of the invention (50 μg/ml) in 22.9 μl of 1×HEPES buffer (50 mM HEPES, pH 7.5, 0.2 M NaCl, 10 mM CaCl₂, 25 μM ZnCl₂ and 0.05% Brij-35) and incubated at room temperature (24° C.) for 2-hr, then 7.1 μl of substrate alpha-2-macroglobulin (0.2 unit/ml) is added and incubated at 37° C. for 20-hr. The reactions are stopped by adding 4× sample buffer and boiled immediately for 5 minutes. After SDS-PAGE, the protein bands are visualized by silver stain.

Synthetic Fluorogenic Peptide Substrates Cleavage Assay

The substrate specificity for polypeptides of the invention with demonstrated metalloproteinase activity can be determined using synthetic fluorogenic peptide substrates (purchased from BACHEM Bioscience Inc). Test substrates include, M-1985, M-2225, M-2105, M-2110, and M-2255. The first four are MMP substrates and the last one is a substrate of tumor necrosis factor-α (TNF-α) converting enzyme (TACE). All the substrates are prepared in 1:1 dimethyl sulfoxide (DMSO) and water. The stock solutions are 50-500 Fluorescent assays are performed by using a Perkin Elmer LS 50B luminescence spectrometer equipped with a constant temperature water bath. The excitation λ is 328 nm and the emission λ is 393 nm. Briefly, the assay is carried out by incubating 176 μl 1×HEPES buffer (0.2 M NaCl, 10 mM CaCl₂, 0.05% Brij-35 and 50 mM HEPES, pH 7.5) with 4 μl of substrate solution (50 μM) at 25° C. for 15 minutes, and then adding 20 μl of a purified polypeptide of the invention into the assay cuvett. The final concentration of substrate is 1 μM. Initial hydrolysis rates are monitored for 30-min.

Example 78 Characterization of the cDNA Contained in a Deposited Plasmid

The size of the cDNA insert contained in a deposited plasmid may be routinely determined using techniques known in the art, such as PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the cDNA sequence. For example, two primers of 17-30 nucleotides derived from each end of the cDNA (i.e., hybridizable to the absolute 5′ nucleotide or the 3′ nucleotide end of the sequence of SEQ ID NO:X, respectively) are synthesized and used to amplify the cDNA using the deposited cDNA plasmid as a template. The polymerase chain reaction is carried out under routine conditions, for instance, in 25 μl of reaction mixture with 0.5 μg of the above cDNA template. A convenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at 55 degree C. for 1 min; elongation at 72 degree C. for 1 min) are performed with a Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis. The PCR product is verified to be the selected sequence by subcloning and sequencing the DNA product. It will be clear that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

Example 79 Cloning, Sequence Analysis and Chromosomal Localization of the Novel Human Integrin Alpha 11 Subunit

The integrins are a large family of cell adhesion molecules consisting of noncovalently associated αβ heterodimers. We have cloned and sequenced a novel human integrin α-subunit cDNA, designated all. The all cDNA encodes a protein with a 22 amino acid signal peptide, a large 1120 residue extracellular domain that contains an I-domain of 207 residues and is linked by a transmembrane domain to a short cytoplasmic domain of 24 amino acids. The deduced a 11 protein shows the typical structural features of integrin α-subunits and is similar to a distinct group of α-subunits from collagen-binding integrins. However, it differs from most integrin α-chains by an incompletetely preserved cytoplasmic GFFKR motif. The human ITGA11 gene was located to bands q22.3-23 on chromosome 15, and its transcripts were found predominantly in bone, cartilage as well as in cardiac and skeletal muscle. Expression of the 5.5 kilobase all mRNA was also detectable in ovary and small intestine.

Introduction

All vertebrate cells express members of the integrin family of cell adhesion molecules, which mediate cellular adhesion to other cells and extracellular subtratum, cell migration and participate in important physiologic processes from signal transduction to cell proliferation and differentiation (Hynes, 92; Springer, 92). Integrins are structurally homologous heterodimeric type-I membrane glycoproteins formed by the noncovalent association of one of eight β-subunits with one of the 17 different α-subunits described to date, resulting in at least 22 different αβ complexes. Their binding specificities for cellular and extracellular ligands are determined by both subunits and are dynamically regulated in a cell-type-specific mode by the cellular environment as well as by the developmental and activation state of the cell (Diamond and Springer, 94). In integrin α-subunits, the aminoterminal region of the large extracellular domain consists of a seven-fold repeated structure which is predicted to fold into a β-propeller domain (Corbi et al., 1987; Springer, 1997). The three or four C-terminal repeats contain putative divalent cation binding motifs that are thought to be important for ligand binding and subunit association (Diamond and Springer, 94). The α¹, α², α¹⁰, α^(D), α^(E), α^(L), α^(M) and α^(X)-subunits contain an approximately 200 amino acid I-domain inserted between the second and third repeat that is not present in other a-chains (Larson et al., 1989). Several isolated I-domains have been shown to independently bind the ligands of the parent integrin heterodimer (Kamata and Takada, 1994; Randi and Hogg, 1994). The α³, α⁵⁻⁸, α^(IIb) and α^(V)-subunits are proteolytically processed at a conserved site into disulphide-linked heavy and light chains, while the α⁴-subunit is cleaved at a more aminoterminal site into two fragments that remain noncovalently associated (Hemler et al., 90). Additional α-subunit variants are generated by alternative splicing of primary transcripts (Ziober et al., 93; Delwel et al., 95; Leung et al., 98). The extracellular domains of α-integrin subunits are connected by a single spanning transmembrane domain to short, diverse cytoplasmic domains whose only conserved feature is a membrane-proximal KXGFF(K/R)R motif (Sastry and Horwitz, 1993). The cytoplasmic domains have been implicated in the cell-type-specific modulation of integrin affinity states (Williams et al., 1994).

Here we report the cDNA cloning, sequence analysis, expression and chromosomal localization of the human α-integrin subunit.

Materials and Methods Library Screening and DNA Sequencing.

A human fetal heart cDNA library in λgt10 (Clontech Laboratories, Inc., Palo Alto, Calif., USA) was screened with ³²P-labelled (REDIPRIME™, Amersham New Zealand Ltd., Auckland, New Zealand) probes corresponding to the regions 473 to 749 and 2394 to 3189 of the all cDNA using standard procedures. Inserts were subcloned from λgt10 into pUC21 and sequenced on both strands according to a successive specific primer strategy on an automated sequencer (Applied Biosystems 373A, The Centre for Gene Technology, School of Biological Sciences, The University of Auckland).

Northern Blot Analysis and Tissue Distribution.

A 1341 bp PCR fragment corresponding to the region 351-1692 of the a cDNA was ³²P-labelled (REDIPRIME™) and hybridized with human multiple tissue Northern blots (MTN I and MTN II, CLONTECH™) for 16 h at 60∞ C. in EXPRESSHYB™ solution (CLONTECH™). Filters were washed twice with 0.1×SSC/1% SDS at 50° C. for 30 min, and autoradiographed. Human DNA from 63 tissue-specific cDNA libraries (Express-Check™, American Type Culture Collection, Manassas, Va., USA) was amplified using primers KL120 (5′-GCAGGGATGCCACCTGCC) and KL119 (5′-GATGAAGACTGTGGTGTCGAAGG) according to the manufacturers instructions. PCR-products were resolved by agarose gel electrophoresis and transferred to Hybond C+ (Amersham). Filters were hybridized by standard procedures (Ausubel et al., 98) with a 502 bp ³²P-labelled (REDIPRIME™) probe fragment obtained from the cloned “¹¹cDNA with the same oligonucleotides.

Chromosomal Assignment.

500 ng genomic DNA prepared from a panel of 21 human-rodent somatic cell hybrids or from human, mouse and hamster cells (Kelsell et al., 95) was amplified with oligonucleotides KL175 (5′-GGTGCCAGACCTACATGGAC) and KL189 (5′-CGTGCAAATTCAATGCCAAATGCC) in a standard PCR reaction of 30 cycles (94° C. for 1 min, 55° C. for 1 min, 72° C. for 2 min). All PCR reactions were resolved in a 2% agarose gel. Southern hybridization was performed as detailed above, except that the probe fragment was obtained from clone HOHBY69 with oligonucleotides KL175 and KL189. For fluorescent in situ hybridization, metaphase spreads were prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes of a 46,XY male donor using standard cytogenetic procedures. A purified 3.7 kB fragment representing the entire coding region of clone HOHBY69 was labelled with biotin-16-dUTP using the High Prime labelling kit (Roche Molecular Biochemicals, Auckland, NZ). Conditions for hybridization and immunofluorescent detection were essentially as described (Morris et al., 93), except that C₀t-1 suppression was not required, slides were washed to a stringency of 0.1×SSC/60° C. after hybridization, and an additional amplification step was needed because of the small size of the probe. For precise chromosome band localization, DAPI and FITC images were captured using a Photometrics KAF1400 CCD camera and QUIPS Smartcapture FISH software version 1.3 (Vysis Inc., Downers Grove, Ill., USA). QUIPS CGH/Karyotyping software (version 3.0.2) assisted karyotype analysis.

Results

Cloning of a Novel Human α-Integrin Subunit cDNA:

A protein homology search (Altschul et al., 90) of the human expressed sequence tag (EST) databases of Human Genome Sciences, Inc. (Ni et al., 97) and The Institute for Genomic Research (Kirkness and Kerlavage, 97) identified the clones HRDAF83 and HOEAM34 as candidate novel integrin α-subunit cDNAs. Clone HRDAF83 was isolated from a human rhabdomyosarcoma cDNA library and sequenced on both strands. The 1223 bp insert contains largely incompletely processed hnRNA and a 277 bp region that showed homology to the aminoterminal half of the α1-integrin I-domain. The 2517 bp insert of clone HOEAM34 was derived from a human osteoblast cDNA library. It is homologous to the C-terminal part of the human α1-subunit and contains 1324 nucleotides of 3′-untranslated region. In order to isolate the full-length cDNAs for these integrin α-subunits, a cDNA library prepared from human fetal heart in λgt10 was screened with the 277 bp fragment from clone HRDAF83 homologous to the α1-I-domain. Two clones, λ831 and λ832, were isolated and both strands of their inserts sequenced. Clone λ832 contains the entire 5′ half of a novel α-subunit cDNA, while clone λ831 covers the same region, but is 358 bp and 173 bp shorter than λ831 at its 5′- and 3′-ends, respectively. A screening of the same library with a 795 bp fragment from the extreme 5′-terminus of clone HOEAM34 identified clone λ342, which contained essentially the same region as clone HOEAM34 but has a 317 bp shorter 3′-untranslated region. Rescreening the EST databases with the sequences derived from the human fetal heart library led to the identification of clone HOHBY69, which was isolated from a osteoblast cDNA libray. Both strands of the 4681 bp insert of clone HOHBY69 were sequenced. The 5′-region of HOHBY69 was identical to the HRDAF83/λ832/λ831-group, while the 3′-region of HOHBY69 was largely identical to HOEAM34 and λ342, thereby demonstrating that the two groups of partial cDNAs represent the 5′- and 3′-portions of the same cDNA. One major difference between the HOHBY69 and HOEAM34/λ342 is the presence of an additional GTA-triplet at position 3088 in HOHBY69. From the overlapping clones, a total of 4986 bp of cDNA was assembled to the composite sequence shown in FIG. 19A-F and has been submitted to GenBank™ with accession number AF109681. This cDNA encodes a previously unidentifed human integrin α-subunit that was designated α11.

Structure of the Human α11-Subunit.

The α11 cDNA contains a 5′-untranslated region of 72 nucleotides and a single open reading frame extending from a predicted translation initiation codon at position +1 to a TGA termination codon at position 3570. This is followed by 1324 nucleotides of 3′ untranslated region which contains an AATTAAA polyadenylation signal (Wahle and Keller, 1996) 12 nucleotides upstream of a poly(A) stretch. The deduced amino acid sequence contains a 22 residue N-terminal region with the characteristics of a cleaved signal peptide (von Heijne, 83; Nielsen et al., 97), a large extracellular domain of 1120 amino acids followed by a 23 amino acid hydrophobic stretch that resembles a transmembrane domain, and a short 24 residue cytoplasmic domain. The molecular weight of the mature 1167 amino acid all-subunit is predicted to be 131 kDa, but the addition of carbohydrate side chains to any of the 15 potential N-glycosylation sequons [NX(S/T)] within the extracellular domain is likely to increase the molecular weight of the native protein. An I-domain of 207 amino acids is inserted between the second and third repeat. Consistent with the structure of an typical 1-domain-containing integrin α-subunit, it lacks a potential dibasic protease cleavage site in the C-terminal region of the extracellular domain.

The α11-subunit is most closely related to the recently discovered α10-subunit (Camper et al., 98, Lehnert et al., in preparation) and the α1- and α2-subunits. Overall, the mature all-protein is 45% identical to the α10 chain, while the homologies to the α1- and α2-subunits are 41% and 39%, respectively. Even greater homology exists between the I-domains of the α10- and α11-subunits which are 60% identical to each other. The high degree of homology seen in the extracellular domains of the subunits is in contrast to the low similarity of their cytoplasmic domains. Interestingly, the KXGFF(K/R)R motif that is absolutely conserved in all other α-subunit cytoplasmic domains is only partially preserved in both subunits. The sequence in α11 is KLGFFRS, while the α10-subunit contains a KLGFFAH motif. A graphical comparison of the similarity between all integrin α-subunits is shown in FIG. 3 (Lehnert et al.). Together with the α-subunits from the collagen-binding integrins α1β1, α2β1 and α10β1, the α11-subunit forms a group distinct from the other 1-domain-containing integrin subunits.

Tissue Distribution and Expression of the Integrin α11-Subunit.

The tissue distribution of the all mRNA was assessed by screening multiple human tissue Northern blots with a probe corresponding to the region 351-1692 of the α11 cDNA. A single transcript of approximately 5.5 kb was found weakly expressed only in ovary and small intestine. Integrin α11-subunit expression was further analyzed by amplification and Southern hybridization of a 502 bp fragment corresponding to the region 1988-2490 in the α11 cDNA from tissue-specific human cDNA libraries. α11 cDNA was detected in five different cDNA libraries prepared from fetal heart (day 57-75), in two fetal brain libraries, and in a cDNA library from large intestine (not shown). An analysis of the Human Genome Sciences Database revealed eight different all-related ESTs in human osteoblast libraries, three EST in a human chondrosarcoma cDNA library and two EST in a human stromal osteoclastoma library.

Chromosomal Localization of the Integrin α11-Subunit.

Genomic DNA from a collection of 21 human-rodent somatic cell hybrids (Kelsell et al., 95) was amplified by PCR using oligonucleotide primers directed the region 473 to 749 of the human α11 cDNA. In Southern hybridization, a signal corresponding to a 1.4 kb fragment was detectable only with DNA from a hybrid cell line that contains human chromosome 15. A fragment of the same size was also amplified from human genomic DNA, but not from mouse or hamster DNA (FIG. 5C (Lehnert et al.)). Cloning and sequence of the PCR product from chromosome 15 revealed the presence of a 1154 bp intron inserted after cDNA-position 600, thus resulting in a PCR-product of 1431 bp. The ITGA11 gene was also localized by fluorescent in situ hybridization of metaphase chromosomes with the entire coding region from clone HOHBY69. All of 20 metaphase cells analyzed showed fluorescent signal on both chromosomes 15, specifically across bands g22.3-q23. No additional signals were detected on any other chromosome (FIG. 5A (Lehnert et al.)).

Discussion:

We have cloned and sequenced a novel cDNA encoding a protein that shares extensive structural homology with integrin α-chains. The aminoterminal 22 amino acids of the deduced protein sequence show the characteristic features of a hydrophobic leader peptide, including a signal peptidase recognition motif at positions −3 and −1 (von Heijne 83). Proteolytic cleavage of the precursor protein at this position would result in an aminoterminal sequence for the mature α11-chain of FNMD, which is similar to the consensus sequence[(F/Y)N(L/V)D] of all other integrin α11-subunits (Tuckwell et al., 94). The N-terminal half of the large extracellular region of all is composed of seven repeats that each contain FG-GAP-GxxY consensus motifs (FG-GAP repeats). These repeats can be found in all integrin α1-subunits and are predicted to fold into a β-propeller domain (Springer, 97). Inserted between the second and third FG-GAP repeats is a 207 amino acid I-domain spanning from glutamine¹³⁸ to methionine³⁴⁴. It contains a divalent cation coordination motif that has been shown to directly bind Mg²⁺ ions in the α^(M) subunit (Michishita et al., 96). The noncontiguous amino acid side chains involved in the coordination of magnesium or manganese ions have been identified by mutagenesis analysis and from crystal structures of the isolated α², α^(L) and α^(M)-subunit I-domains (Emsley et al., 97; Qu and Leahy, 95; Lee et al., 95). All residues required for the coordination of the divalent cations in these subunits are preserved in the α11-I-domain. These are the asparagines at positions 148 and 249, the serine residues at position 150 and 152, and the threonine at position 218.

The crystal sctructure of the α2-subunit has revealed a small “-helix that is not present in the I-domains of the β2-associated α-subunits. Together with the MIDAS sphere, amino acid residues from this C-helix and the adjacent turn region have been proposed to make physical contacts to a collagen triple helix (Emsley et al., 97). Interestingly, the small C-helix is structurally conserved in the α-subunits of the collagen-binding integrins α1β1, α2β1 and α10β1, and is also present in the α11 I-domain (G²⁷⁹YYNR²⁸³). In addition, asparagine¹⁵⁴ and histidine²⁵⁸ of the α2-I-domain were predicted to contact the collagen triple helix, and both are preserved in the α1, α10 and α11-I-domains, but not in other integrin all-subunits. The conservation of structural motifs required for collagen binding suggests that collagen may be a ligand for the α11 integrin. Each of the repeats 5-7 of the α11-subunit accommodates the sequence Dx(D/N)xDxxxD. Three or four copies of these putative divalent cation binding sites are conserved in all integrin α-subunits and their presence is consistent with the divalent cation requirement for the adhesive function of integrins (Larson et al., 89; Fujimura and Phillips, 83; Hynes, 92). The extracellular domain of the integrin α11-subunit contains 20 cysteine residues. Only the intramolecular disulfide bonds in the “^(IIb)subunit have been biochemically characterized (Calvete et al., 89), but the location of many cysteines is conserved in integrin α-subunits. In the α11-subunit, the cysteine residues 637 and 646, 652 and 707, 759 and 765, and 859 and 871 are homologous to the residues that form the four carboxyterminal disulfide bonds in the heavy chain of “^(IIb)(Calvete et al., 89). Based on the proposed structure of the integrin α-subunit propeller domain (Springer et al., 97), additional disulfide bonds within the α11 subunit can be predicted between cysteine residues 54 and 61, 99 and 117, and between 107 and 137. Two additional cysteine residues are found within a short segment (residues 783 to 798) that is unique to the α11-subunit.

The integrin cytoplasmic domains play central roles in integrin affinity modulation and in cellular signal transmission. The membrane-proximal sequence KxGFF(K/R)R is strictly conserved among integrin α-subunit cytoplasmic domains (Williams et al., 94). Within this motif, both phenylalanine residues and the last arginine have been implicated in maintaining the default low affinity state of integrins α^(L)β₂ and α^(IIb)β₃, as their substitution or deletion resulted in constitutively activated ligand binding (O'Toole et al., 94, Lu and Spriner 97). Interestingly, the last arginine residue is replaced by a serine in the all cytoplasmic domain and with a histidine in the α10 subunit, suggesting that both integrins might be in a default “high” affinity state. It will be interesting to analyze whether substitution of these residues with a conserved arginine will affect their affinity status.

We have isolated α11 cDNAs from osteoclast, osteoblast, myosarcoma and fetal heart libraries. Amongst the HGS EST databases, integrin all transcripts were predominantly found in libraries prepared from osteoblast, osteoclast and chondrosarcoma cells. A search for further “¹¹-related sequences in the EST division of the GenBank database revealed two clones (accession numbers Z50157 and Z50167) from primary human myoblasts (Genini et al., 96), two clones from human trabecular bone cells (AA852614 and AA852615), as well as clones from fibroblast cells (W45078), pancreatic tumor (U53091) and breast tissue (H16112). In contrast, Northern blot analysis detected α11-expression only in ovary and small intestine. only fetal heart, fetal brain and large intestine. Of the tissues represented in the tissue-specific cDNA-library panel, only fetal heart, fetal brain and large intestine showed detectable α11-expression. However, bone- and muscle-derived tissues were not included in the Northern blot, and cDNA libraries prepared from these tissues were also not represented in the tissue-specific cDNA panel.

The ITGA11gene was localized to chromosome 15, bands q22.3-23, by FISH and PCR analysis of human-rodent somatic cell hybrids. This segment is overrepresented in squamous cell carcinomas (Wolff et al., 1998), but appears to only infrequently affected in other cancers. Genes at this region encode neogenin, a protein expressed ubiquitously expressed in human tissues (Meyerhardt et al., 97); tropomyosin 1, expressed in cardiac and skeletal muscle tissues (Tiso et al., 97); and the human homologue of the metalloprotease-disintegrin kuzbanian, which is overexpressed in tumors of sympathoadrenal origin (Yavari et al., 98). In addition, the region 15q22.3-q23 is linked to Bardet-Biedl syndrome 4, a heterogeneous autosomal disorder characterized by obesity and associated with cardiovascular anomalities (Carmi et al., 95).

In conclusion, we have cloned and sequenced the cDNA for the novel integrin α11-subunit which is closely related to the “-subunits of the collagen-binding integrins α1β1, α2β1 and α10β1. The high degree of homology of α11 to these subunits suggests that it associates with the integrin β1-subunit, and may function as an additional collagen receptor.

All references referred to above and presented below are hereby incorporated herein by reference:

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TABLE 8 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 A . . . . . . 1.30 −0.61 . . . 0.95 2.10 Arg 2 . . B . . . . 0.88 −1.04 * . . 0.95 3.21 Lys 3 . . B B . . . 0.98 −0.79 * . . 0.75 2.07 Thr 4 A . . B . . . 1.02 −0.30 . . . 0.45 2.20 Arg 5 . . B B . . . 0.60 −0.49 . . F 0.60 1.11 Leu 6 A . . B . . . 0.39 0.20 * . . −0.30 0.46 Trp 7 . . B B . . . −0.01 0.89 * . . −0.60 0.26 Gly 8 A . . B . . . −0.66 1.31 . * . −0.60 0.14 Leu 9 A . . B . . . −1.16 1.93 * . . −0.60 0.17 Leu 10 . . B B . . . −1.97 1.93 * * . −0.60 0.13 Trp 11 . . B B . . . −2.01 1.80 . . . −0.60 0.12 Met 12 . . B B . . . −2.02 2.01 . . . −0.60 0.10 Leu 13 A . . B . . . −1.68 1.71 . . . −0.60 0.17 Phe 14 A . . B . . . −1.68 1.03 * * . −0.60 0.28 Val 15 A . . B . . . −0.76 0.80 * * . −0.60 0.23 Ser 16 A A . . . . . −1.06 0.19 * * . −0.30 0.55 Glu 17 A A . . . . . −1.04 0.00 . * . −0.30 0.64 Leu 18 A A . . . . . −0.54 −0.29 * * . 0.30 0.88 Arg 19 A A . . . . . 0.20 −0.44 * * . 0.30 0.94 Ala 20 A A . . . . . 0.24 −0.83 * * . 0.75 1.09 Ala 21 A A . . . . . 0.23 −0.14 * * . 0.45 1.09 Thr 22 A A . . . . . 0.23 −0.34 * * F 0.45 0.80 Lys 23 A A . . . . . 1.04 −0.34 * * F 0.60 1.38 Leu 24 A A . . . . . 0.98 −0.84 * * F 0.90 2.36 Thr 25 A A . . . . . 1.32 −1.34 . . F 0.90 3.27 Glu 26 A A . . . . . 1.91 −1.07 . . F 0.90 2.56 Glu 27 A A . . . . . 1.41 −1.07 . * F 0.90 5.38 Lys 28 A A . . . . . 1.41 −1.07 . * F 0.90 3.08 Tyr 29 A A . . . . . 2.22 −1.56 . * F 0.90 3.55 Glu 30 A A . . . . . 2.19 −1.56 . * F 0.90 3.55 Leu 31 A A . . . . . 2.19 −1.13 . * F 0.90 1.76 Lys 32 A A . . . . . 1.88 −0.73 * * F 0.90 1.94 Glu 33 A A . . . . . 1.02 −1.00 * * F 0.90 1.62 Gly 34 A A . . . . . 1.27 −0.31 . * F 0.60 1.62 Gln 35 A A . . . . . 0.41 −1.00 . * F 0.90 1.35 Thr 36 A A . . . . . 1.27 −0.36 . * F 0.45 0.58 Leu 37 A A . . . . . 0.56 −0.36 . * F 0.60 1.17 Asp 38 . A B . . . . 0.56 −0.21 . * . 0.30 0.36 Val 39 . A B . . . . 0.66 −0.61 . * . 0.60 0.42 Lys 40 . A B . . . . 0.34 −0.34 . * . 0.30 0.80 Cys 41 . . B . . T . −0.16 −0.54 . * . 1.00 0.69 Asp 42 A . . . . T . 0.66 0.14 . * . 0.10 0.77 Tyr 43 A . . . . T . 0.70 −0.50 . * . 0.70 0.66 Thr 44 A . . . . T . 0.86 −0.50 * * . 0.85 2.47 Leu 45 A A . . . . . 0.22 −0.29 * * . 0.45 1.28 Glu 46 A A . . . . . 0.59 0.21 * . . −0.30 0.83 Lys 47 A A . . . . . 0.29 −0.16 . . . 0.30 0.77 Phe 48 A A . . . . . 0.53 −0.26 * . F 0.60 1.25 Ala 49 A . . . . T . 0.89 −0.54 . . F 1.30 1.25 Ser 50 A . . . . T . 1.11 −0.54 * . F 1.30 1.25 Ser 51 A . . . . T . 0.82 −0.04 . . F 1.00 1.46 Gln 52 A . . . . T . 0.78 0.09 . * F 0.40 1.51 Lys 53 A . . . . . . 0.59 −0.01 * . F 0.80 1.96 Ala 54 A . . B . . . 0.29 0.29 * . . −0.15 1.02 Trp 55 A . . B . . . 0.70 0.59 * . . −0.60 0.41 Gln 56 . . B B . . . 1.00 0.19 * . . −0.30 0.41 Ile 57 . . B B . . . 0.66 0.19 * . . 0.04 0.67 Ile 58 . . B . . T . 0.61 0.11 * . . 0.78 0.63 Arg 59 . . B . . T . 0.60 −0.80 * . . 2.02 0.63 Asp 60 . . . . T T . 0.68 −0.59 * . F 2.91 0.89 Gly 61 . . . . T T . 0.72 −0.84 * . F 3.40 1.97 Glu 62 . . . . . . C 1.30 −1.53 * . F 2.66 2.01 Met 63 . . . . . T C 1.38 −1.04 . . F 2.52 1.74 Pro 64 . . . . T T . 0.68 −0.36 * . F 2.08 1.45 Lys 65 . . . . T T . 0.01 −0.29 . . F 1.59 0.84 Thr 66 A . . . . T . 0.04 0.29 . . F 0.25 0.46 Leu 67 A A . . . . . 0.04 0.16 * . . −0.30 0.43 Ala 68 A A . . . . . 0.76 −0.27 * . . 0.30 0.37 Cys 69 A A . . . . . 0.76 −0.27 * . . 0.30 0.50 Thr 70 A A . . . . . 0.41 −0.33 * . . 0.64 0.94 Glu 71 A . . . . . . 0.77 −0.63 * . F 1.78 1.25 Arg 72 A . . . . . . 1.58 −1.13 * . F 2.12 4.65 Pro 73 . . . . T . . 1.87 −1.30 . . F 2.86 5.18 Ser 74 . . . . T T . 2.50 −1.40 * . F 3.40 4.01 Lys 75 . . . . T T . 2.60 −0.90 * . F 3.06 2.79 Asn 76 . . . . T T . 1.74 −0.47 * . F 2.42 2.79 Ser 77 . . . . . T C 1.63 −0.26 . * F 1.88 1.54 His 78 . . B B . . . 0.99 −0.24 . . F 0.94 1.34 Pro 79 . . B B . . . 0.94 0.40 . . . −0.60 0.62 Val 80 . . B B . . . 1.01 0.43 . * . −0.60 0.46 Gln 81 . . B B . . . 0.12 0.04 * . . −0.30 0.66 Val 82 . . B B . . . −0.47 0.23 * . . −0.30 0.30 Gly 83 . . B B . . . −1.24 0.49 * * . −0.60 0.28 Arg 84 . . B B . . . −1.03 0.53 * . . −0.60 0.13 Ile 85 . . B B . . . −0.18 0.13 * . . −0.30 0.31 Ile 86 . . B B . . . −0.42 −0.51 * . . 0.60 0.53 Leu 87 . . B B . . . 0.40 −0.19 * * . 0.30 0.42 Glu 88 . . B B . . . 0.74 0.31 * * . −0.30 0.82 Asp 89 A . . . . . . 0.60 −0.37 * * . 0.65 1.95 Tyr 90 A . . . . . . 1.14 −0.56 . . . 0.95 3.21 His 91 A . . . . T . 1.22 −0.81 . . . 1.15 1.84 Asp 92 A . . . . T . 1.22 −0.13 . * . 0.70 0.91 His 93 A . . . . T . 1.33 0.56 * * . −0.20 0.48 Gly 94 A . . . . T . 0.48 −0.20 . * . 0.70 0.69 Leu 95 A . . B . . . 0.83 −0.06 . * . 0.30 0.31 Leu 96 A . . B . . . 0.27 −0.06 . * . 0.30 0.44 Arg 97 . . B B . . . −0.59 0.06 . * . −0.30 0.44 Val 98 . . B B . . . −0.56 0.27 . * . −0.30 0.40 Arg 99 . . B B . . . −1.02 −0.01 . * . 0.30 0.77 Met 100 . . B B . . . −0.21 −0.01 . * . 0.30 0.32 Val 101 . . B B . . . −0.26 0.39 . * . −0.30 0.76 Asn 102 . . B B . . . −0.37 0.39 . * . −0.30 0.29 Leu 103 . . B B . . . 0.49 0.39 * * . −0.30 0.50 Gln 104 . . B B . . . 0.08 −0.23 * * . 0.73 1.13 Val 105 . . B B . . . 0.33 −0.49 . * . 0.86 0.94 Glu 106 . . B B . . . 0.38 −0.46 . * F 1.44 1.13 Asp 107 . . . . T T . 0.13 −0.46 . * F 2.37 0.54 Ser 108 . . . . T T . 0.94 −0.10 . * F 2.80 1.14 Gly 109 . . . . T T . 0.28 −0.34 . . F 2.52 1.14 Leu 110 . . . . T T . 0.28 0.23 . . . 1.34 0.36 Tyr 111 . . B B . . . −0.61 0.87 . . . −0.04 0.20 Gln 112 . . B B . . . −0.86 1.17 . . . −0.32 0.14 Cys 113 . . B B . . . −0.56 1.50 . . . −0.60 0.27 Val 114 . . B B . . . −0.42 1.21 . . . −0.60 0.30 Ile 115 . . B B . . . 0.18 0.89 * . . −0.26 0.27 Tyr 116 . . B . . . . 0.47 0.91 * . . 0.28 0.77 Gln 117 . . . . . . C 0.47 0.34 * . . 1.27 2.09 Pro 118 . . . . . T C 0.92 −0.30 * . F 2.56 5.15 Pro 119 . . . . T T . 1.74 −0.56 * . F 3.40 5.08 Lys 120 . . . . . T C 2.03 −0.81 * . F 2.86 3.99 Glu 121 . . . . . T C 1.47 −0.60 * . F 2.52 2.56 Pro 122 A . . . . . . 0.77 −0.34 * . F 1.48 1.36 His 123 A . . B . . . 0.98 0.01 * . . 0.04 0.59 Met 124 A . . B . . . 1.30 0.01 * . . −0.30 0.57 Leu 125 A . . B . . . 0.37 0.01 * * . −0.30 0.72 Phe 126 A . . B . . . 0.48 0.27 * * . −0.30 0.37 Asp 127 A . . B . . . −0.12 −0.23 * * . 0.30 0.74 Arg 128 A . . B . . . −0.94 −0.16 * * . 0.30 0.74 Ile 129 A . . B . . . −1.20 −0.20 * * . 0.30 0.63 Arg 130 . . B B . . . −0.70 −0.34 * * . 0.30 0.28 Leu 131 . . B B . . . 0.04 0.14 * * . −0.30 0.21 Val 132 . . B B . . . −0.30 0.14 * * . −0.30 0.59 Val 133 . . B B . . . −1.11 −0.11 * * . 0.30 0.30 Thr 134 . . B B . . . −0.52 0.67 * * F −0.45 0.31 Lys 135 . . B B . . . −0.98 0.37 . * F −0.15 0.56 Gly 136 . . B . . T . −0.48 0.16 . . F 0.25 0.75 Phe 137 . . B . . T . 0.17 0.00 . . F 0.25 0.75 Ser 138 . . B . . T . 0.68 −0.06 . . F 0.85 0.58 Gly 139 . . . . . T C 0.69 0.37 . . F 0.45 0.58 Thr 140 . . . . . T C 0.64 0.33 . . F 0.45 0.90 Pro 141 . . . . . T C 0.99 −0.06 . . F 1.20 1.08 Gly 142 . . . . . T C 1.69 −0.44 . . F 1.54 1.89 Ser 143 . . . . . T C 1.69 −0.47 . . F 1.88 2.11 Asn 144 . . . . . T C 1.72 −0.57 . . F 2.52 1.82 Glu 145 . . . . . T C 2.03 −0.51 . . F 2.86 2.66 Asn 146 . . . . T T . 2.24 −0.54 . . F 3.40 3.44 Ser 147 . . . . T T . 1.73 −0.53 . . F 3.06 3.44 Thr 148 . . . B T . . 1.79 −0.29 * . F 2.02 1.47 Gln 149 . . B B . . . 1.83 0.47 * . F 0.38 1.44 Asn 150 . . B B . . . 0.94 0.07 * . F 0.34 2.14 Val 151 . . B B . . . 0.73 0.37 * . F 0.00 1.04 Tyr 152 . . B B . . . 0.82 0.31 * . . −0.30 0.93 Lys 153 . . B B . . . 0.82 0.34 * . F −0.15 0.89 Ile 154 . . B B . . . 0.51 0.43 * . F −0.30 1.74 Pro 155 . . B . . T . 0.20 0.27 * . F 0.40 1.60 Pro 156 . . . . T T . 1.10 0.00 * . F 0.80 1.15 Thr 157 . . . . T T . 0.76 0.00 * . F 0.80 3.29 Thr 158 . . B . . T . −0.10 −0.19 * . F 1.00 2.15 Thr 159 . A B . . . . 0.12 0.07 * . F 0.00 1.15 Lys 160 . A B . . . . 0.12 0.21 * . F −0.15 0.43 Ala 161 . A B . . . . −0.48 0.16 * . . −0.30 0.46 Leu 162 . A B . . . . −0.41 0.36 * . . −0.30 0.26 Cys 163 . . B . . T . −0.41 0.63 * . . −0.20 0.20 Pro 164 . . B . . T . −0.40 1.11 * . . −0.20 0.29 Leu 165 . . B . . T . −0.66 1.00 * * . −0.20 0.47 Tyr 166 . . B . . T . 0.04 0.74 * * . −0.05 1.37 Thr 167 . . B . . . . 0.54 0.17 * . F 0.20 1.74 Ser 168 . . . . . T C 0.36 0.23 * . F 0.60 3.04 Pro 169 . . B . . T . 0.26 0.19 * . F 0.40 1.44 Arg 170 . . B . . T . 1.07 −0.09 * * F 1.00 1.44 Thr 171 . . B . . T . 0.72 −0.17 * . F 1.00 1.86 Val 172 . . B B . . . 0.82 −0.06 * . F 0.60 1.21 Thr 173 . . B B . . . 0.91 −0.06 * * F 0.73 0.96 Gln 174 . . B B . . . 1.17 0.37 * * F 0.56 1.03 Ala 175 . . B B . . . 0.76 −0.11 * * F 1.44 2.77 Pro 176 . . . . . T C 0.76 −0.37 . . F 2.32 2.57 Pro 177 . . . . T T . 1.02 −0.37 . * F 2.80 2.14 Lys 178 . . . . T T . 1.33 −0.27 . * F 2.52 2.14 Ser 179 . . B . . T . 0.48 −0.77 . * F 2.14 2.31 Thr 180 . . B B . . . 0.77 −0.56 . * F 1.46 1.11 Ala 181 . . B B . . . 0.67 −0.60 . * F 1.03 0.74 Asp 182 . . B B . . . 0.67 −0.11 . * F 0.45 0.80 Val 183 . . B B . . . 0.62 −0.07 . * F 0.79 0.86 Ser 184 . . B . . . . 0.62 −0.56 . . F 1.78 1.42 Thr 185 . . B . . T . 0.93 −0.67 . * F 2.32 1.14 Pro 186 . . . . . T C 0.63 −0.67 . * F 2.86 2.66 Asp 187 . . . . T T . 0.63 −0.63 . * F 3.40 1.39 Ser 188 . . B . . T . 0.68 −0.61 . * F 2.66 1.55 Glu 189 . . B . . . . 0.67 −0.41 . * F 1.67 0.83 Ile 190 . . B . . . . 0.98 −0.36 . * F 1.33 0.71 Asn 191 . . B . . . . 0.33 0.04 . * . 0.24 0.86 Leu 192 . . B B . . . 0.02 0.30 . * . −0.30 0.37 Thr 193 . . B B . . . 0.32 0.79 . * F −0.60 0.76 Asn 194 . . B B . . . −0.57 0.10 * * F −0.15 0.78 Val 195 . . B B . . . −0.57 0.39 * * F −0.15 0.67 Thr 196 . . B B . . . −0.46 0.39 * * F −0.15 0.32 Asp 197 . . B B . . . −0.50 −0.10 * * . 0.30 0.39 Ile 198 . . B B . . . −0.40 0.14 * * . −0.30 0.39 Ile 199 . . B B . . . −1.26 −0.07 * * . 0.30 0.42 Arg 200 . . B B . . . −1.10 0.09 * * . −0.30 0.19 Val 201 . . B B . . . −0.79 0.87 * . . −0.60 0.23 Pro 202 . . B B . . . −1.68 0.59 * . . −0.60 0.53 Val 203 . . B B . . . −1.64 0.59 . * . −0.60 0.19 Phe 204 . . B B . . . −1.64 1.23 . * . −0.60 0.19 Asn 205 . . B B . . . −2.57 1.27 . * . −0.60 0.09 Ile 206 . . B B . . . −2.52 1.53 . . . −0.60 0.10 Val 207 . . B B . . . −2.90 1.57 . . . −0.60 0.09 Ile 208 . . B B . . . −2.39 1.29 . . . −0.60 0.06 Leu 209 . . B B . . . −2.03 1.31 . * . −0.60 0.08 Leu 210 . . B . . T . −2.73 1.06 . . . −0.20 0.11 Ala 211 . . B . . T . −2.66 1.20 . . . −0.20 0.13 Gly 212 A . . . . T . −2.10 1.20 . . . −0.20 0.13 Gly 213 A . . . . T . −1.17 0.90 . . . −0.20 0.22 Phe 214 A . . . . . . −0.66 0.21 . . . −0.10 0.43 Leu 215 A . . . . . . −0.66 0.10 . . F 0.05 0.58 Ser 216 . . B . . T . −0.92 0.36 . . F 0.25 0.48 Lys 217 . . B . . T . −1.28 0.57 . . F −0.05 0.42 Ser 218 . . B . . T . −1.23 0.57 . . F −0.05 0.44 Leu 219 . . B . . T . −1.39 0.27 . . . 0.10 0.44 Val 220 . . B B . . . −1.39 0.53 . . . −0.60 0.16 Phe 221 . . B B . . . −1.79 1.21 . . . −0.60 0.10 Ser 222 . . B B . . . −2.42 1.61 . . . −0.60 0.10 Val 223 . . B B . . . −2.98 1.43 . . . −0.60 0.14 Leu 224 . . B B . . . −2.48 1.43 . * . −0.60 0.12 Phe 225 A . . B . . . −2.43 1.13 * * . −0.60 0.13 Ala 226 A . . B . . . −1.62 1.43 * * . −0.60 0.15 Val 227 A . . B . . . −1.62 0.79 * * . −0.60 0.35 Thr 228 . . B B . . . −1.47 0.49 . * . −0.60 0.54 Leu 229 . . B B . . . −1.51 0.49 * * . −0.60 0.46 Arg 230 . . B B . . . −1.02 0.63 * * . −0.60 0.46 Ser 231 . . B B . . . −0.82 0.41 . * . −0.60 0.49 Phe 232 . . B B . . . −0.36 0.36 . * . −0.30 0.76 Val 233 . . B B . . . −0.43 0.10 * * . −0.30 0.50 Pro 234 . . B B . . . −0.01 0.53 * * . −0.60 0.48

TABLE 9 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 A A . . . . . 0.61 −0.60 . . . 0.75 1.47 Lys 2 A A . . . . . 0.41 −0.64 . . . 0.96 1.54 Arg 3 . A B . . . . 0.46 −0.57 * . . 1.17 1.22 Ala 4 A A . . . . . 0.50 −0.57 * . . 1.38 1.22 Ser 5 A . . . . T . 0.59 −0.76 * . F 1.99 0.60 Ala 6 . . . . . T C 1.30 −0.37 * . F 2.10 0.41 Gly 7 . . . . . T C 0.44 −0.37 * . F 1.89 0.80 Gly 8 . . . . . T C −0.48 −0.19 * * F 1.68 0.49 Ser 9 . A . . . . C −0.48 0.11 . . F 0.47 0.40 Arg 10 . A B . . . . −0.47 0.11 * . F 0.06 0.41 Leu 11 . A B . . . . −0.73 0.60 * . . −0.60 0.43 Leu 12 . A B . . . . −1.20 0.81 * . . −0.60 0.24 Ala 13 . A B . . . . −1.14 1.11 * . . −0.60 0.10 Trp 14 . A B . . . . −1.66 2.03 . * . −0.60 0.13 Val 15 . A B . . . . −1.77 2.03 . * . −0.60 0.13 Leu 16 A A . . . . . −1.54 1.74 . . . −0.60 0.22 Trp 17 A A . . . . . −1.02 1.74 . . . −0.60 0.21 Leu 18 A A . . . . . −0.43 1.74 . . . −0.60 0.30 Gln 19 A A . . . . . −1.00 1.50 . . . −0.60 0.63 Ala 20 . A . . T . . −0.73 1.46 . . . −0.20 0.45 Trp 21 . A . . T . . −0.51 1.04 . . . −0.20 0.55 Gln 22 . A B . . . . −0.43 0.86 . . . −0.60 0.32 Val 23 . A B . . . . −0.29 0.89 . . . −0.60 0.49 Ala 24 . A B . . . . −0.50 0.96 . . . −0.60 0.25 Ala 25 . A B . . . . −0.26 0.47 . . . −0.60 0.22 Pro 26 . A . . T . . −0.56 0.50 . * . −0.20 0.30 Cys 27 . . . . T T . −1.22 0.36 . . . 0.50 0.30 Pro 28 . . . . T T . −1.22 0.43 . . . 0.20 0.16 Gly 29 . . . . T T . −1.30 0.57 . . . 0.20 0.08 Ala 30 . . B . . T . −0.96 0.71 . . . −0.20 0.08 Cys 31 . . B . . . . −0.74 0.90 . . . −0.40 0.08 Val 32 . . B . . . . −0.08 0.87 . . . −0.40 0.12 Cys 33 . . B . . T . −0.08 0.44 . . . −0.20 0.21 Tyr 34 . . B . . T . 0.31 0.37 . . . 0.36 0.62 Asn 35 . . B . . T . 0.04 −0.20 . . F 1.52 1.66 Glu 36 . . B . . T . 0.40 −0.20 . . F 1.78 2.30 Pro 37 . . . B T . . 0.94 −0.29 . . F 2.04 2.11 Lys 38 . . . B T . . 1.31 −0.56 . * F 2.60 1.90 Val 39 . . . B T . . 0.89 −0.57 . * F 2.34 1.47 Thr 40 . . B B . . . 0.68 0.00 . * F 0.63 0.51 Thr 41 . . B B . . . 0.68 0.00 . * F 0.37 0.39 Ser 42 . . B B . . . 0.89 0.40 * * F −0.19 0.92 Cys 43 . . B . . T . 0.50 0.16 * * F 0.40 1.10 Pro 44 . . . . T T . 0.54 0.10 . . F 0.65 0.76 Gln 45 . . . . T T . 0.86 0.30 . . F 0.65 0.47 Gln 46 . . B . . T . 0.58 0.31 * . F 0.40 1.50 Gly 47 . A B . . . . 0.02 0.24 * . F −0.15 0.98 Leu 48 . A B B . . . 0.48 0.46 . . . −0.60 0.42 Gln 49 . A B B . . . −0.17 0.49 . . . −0.60 0.38 Ala 50 . A B B . . . −0.51 0.73 * . . −0.60 0.28 Val 51 . A B B . . . −1.40 0.73 * . . −0.60 0.34 Pro 52 . . B B . . . −1.27 0.73 . . . −0.60 0.14 Val 53 . . B B . . . −1.04 0.76 . . . −0.60 0.21 Gly 54 . . B . . . . −1.63 0.76 . . . −0.40 0.29 Ile 55 . . B . . . . −1.34 0.61 . . . −0.40 0.19 Pro 56 . . B . . . . −0.49 0.57 . * . −0.40 0.34 Ala 57 A . . . . . . −0.17 0.33 . * . −0.10 0.59 Ala 58 A . . . . . . −0.20 −0.10 * . . 0.65 1.64 Ser 59 A . . B . . . −0.56 −0.10 * * F 0.45 0.75 Gln 60 . . B B . . . −0.48 0.26 * * F −0.15 0.64 Arg 61 . . B B . . . −0.30 0.44 . * F −0.45 0.52 Ile 62 . . B B . . . −0.06 0.44 . * . −0.60 0.53 Phe 63 . . B B . . . 0.53 0.49 * * . −0.60 0.30 Leu 64 . . B B . . . 0.94 0.49 * * . −0.60 0.25 His 65 . . . . T T . 0.06 0.49 * * . 0.26 0.69 Gly 66 . . . . T T . −0.36 0.49 * * F 0.47 0.56 Asn 67 . . . . T T . 0.50 0.09 * . F 0.83 0.91 Arg 68 . . . . T T . 0.34 −0.10 . . F 1.49 0.91 Ile 69 . . . . T . . 0.94 0.04 . . . 0.60 0.69 Ser 70 . . B . . . . 0.39 0.04 * . . 0.14 0.66 His 71 . . B . . . . 0.14 0.14 * . . 0.08 0.34 Val 72 . . B . . . . −0.16 0.64 * . . −0.28 0.49 Pro 73 . . B . . . . −0.97 0.34 * * . −0.04 0.49 Ala 74 . . . . T . . 0.03 0.74 * * . 0.00 0.31 Ala 75 A . . . . . . −0.26 0.24 . * . −0.10 0.82 Ser 76 A . . . . . . −0.89 0.10 * . . −0.10 0.54 Phe 77 A . . . . . . 0.08 0.24 * . . −0.10 0.29 Arg 78 A . . . . . . 0.29 −0.26 * * . 0.60 0.55 Ala 79 A . . . . . . 0.07 −0.36 * * . 0.70 0.66 Cys 80 A . . . . T . 0.34 −0.06 * * . 1.00 0.63 Arg 81 . . . . T T . −0.24 −0.36 * * . 1.50 0.47 Asn 82 . . . . T T . −0.36 0.33 * * . 1.00 0.32 Leu 83 . . B . . T . −0.76 0.51 * . . 0.20 0.50 Thr 84 . . B B . . . −0.98 0.86 * . . −0.30 0.27 Ile 85 . . B B . . . −0.34 1.54 . . . −0.40 0.14 Leu 86 . . B B . . . −0.76 1.64 . . . −0.50 0.23 Trp 87 . . B B . . . −0.76 1.34 . * . −0.60 0.21 Leu 88 . . B B . . . −0.80 1.26 . . . −0.60 0.48 His 89 . . B . . T . −1.30 1.21 . . . −0.20 0.43 Ser 90 . . . . . T C −1.00 1.21 * * . 0.00 0.34 Asn 91 . . . . . T C −0.08 0.80 * * . 0.00 0.42 Val 92 A . . . . T . −0.68 0.11 . * . 0.10 0.60 Leu 93 A A . . . . . 0.13 0.30 . * . −0.30 0.31 Ala 94 A A . . . . . −0.42 −0.09 . * . 0.30 0.33 Arg 95 A A . . . . . −0.71 0.01 * * . −0.30 0.44 Ile 96 A A . . . . . −1.30 −0.13 * * . 0.30 0.54 Asp 97 A A . . . . . −1.14 −0.31 . * . 0.30 0.54 Ala 98 A A . . . . . −0.64 −0.03 * * . 0.30 0.24 Ala 99 A A . . . . . −0.40 0.46 * * . −0.60 0.49 Ala 100 A A . . . . . −1.32 0.20 . * . −0.30 0.29 Phe 101 A A . . . . . −1.02 0.89 . . . −0.60 0.24 Thr 102 A A . . . . . −1.83 0.89 . . . −0.60 0.24 Gly 103 A A . . . . . −2.06 1.07 . . . −0.60 0.19 Leu 104 A A . . . . . −1.47 1.26 . . . −0.60 0.19 Ala 105 A A . . . . . −0.88 0.47 . . . −0.60 0.22 Leu 106 A A . . . . . −0.99 0.39 . . . −0.30 0.39 Leu 107 A A . . . . . −0.68 0.64 . * . −0.60 0.39 Glu 108 A A . . . . . −1.14 −0.04 . * . 0.30 0.64 Gln 109 A A . . . . . −0.63 0.14 . * . −0.30 0.64 Leu 110 A A . . . . . −0.04 −0.16 . * . 0.45 1.05 Asp 111 A A . . . . . 0.77 −0.84 . * . 0.75 1.01 Leu 112 A A . . . . . 0.99 −0.44 . * . 0.30 0.94 Ser 113 A . . . . T . 0.99 −0.34 . * F 1.00 1.15 Asp 114 A . . . . T . 0.18 −0.63 . * F 1.30 1.19 Asn 115 A . . . . T . 1.10 0.06 . * F 0.40 1.19 Ala 116 A . . . . T . 0.80 −0.63 . * . 1.15 1.74 Gln 117 . A B . . . . 0.76 −0.63 . * . 0.75 1.40 Leu 118 . A B . . . . 1.06 0.01 . * . −0.30 0.64 Arg 119 . A B . . . . 0.84 −0.39 * * F 0.60 1.07 Ser 120 . A B . . . . 0.26 −0.46 * * F 0.53 0.95 Val 121 . . B . . . . 0.53 −0.36 . * F 0.96 1.17 Asp 122 . . B . . T . −0.17 −0.56 . * F 1.39 0.86 Pro 123 . . B . . T . 0.61 0.23 * * F 0.57 0.55 Ala 124 . . B . . T . 0.16 0.34 * . F 0.80 1.02 Thr 125 . . B . . T . −0.36 0.13 . . . 0.42 0.60 Phe 126 . . B . . . . 0.16 0.81 . * . −0.16 0.32 His 127 A . . . . . . 0.27 0.81 * * . −0.24 0.31 Gly 128 . . . . . . C −0.33 0.31 * * . 0.18 0.43 Leu 129 . A . . . . C 0.22 0.51 * * . −0.40 0.41 Gly 130 . A . . . . C 0.22 0.23 * * . −0.10 0.41 Arg 131 A A . . . . . 0.11 0.21 * * . −0.30 0.59 Leu 132 A A . . . . . 0.11 0.47 . * . −0.60 0.59 His 133 . A B . . . . −0.36 0.29 * * . −0.30 0.82 Thr 134 . A B . . . . 0.46 0.54 * . . −0.60 0.34 Leu 135 . A B . . . . 0.91 0.54 * * . −0.38 0.70 His 136 . A B . . . . 0.13 −0.14 * . . 0.89 1.00 Leu 137 . A B . . . . 0.60 −0.07 . . . 0.96 0.37 Asp 138 . . . . T T . −0.18 −0.13 * . . 1.98 0.45 Arg 139 . . . . T T . 0.13 −0.13 . . . 2.20 0.27 Cys 140 . . . . T T . 0.94 −0.23 * . . 1.98 0.57 Gly 141 . . B . . T . 0.17 −0.91 * . . 1.66 0.59 Leu 142 . A B . . . . 0.63 −0.23 * . . 0.74 0.25 Gln 143 . A B . . . . 0.42 0.20 * . . −0.08 0.46 Glu 144 . A B . . . . −0.03 0.06 * * F −0.15 0.72 Leu 145 . A B . . . . −0.18 0.06 * . F −0.15 0.86 Gly 146 . . . . . T C −0.53 0.06 * * F 0.45 0.41 Pro 147 . . . . T T . 0.39 0.44 * * F 0.35 0.20 Gly 148 . . . . . T C 0.04 0.44 * . F 0.15 0.49 Leu 149 . . B . . T . −0.77 0.19 * * . 0.10 0.49 Phe 150 . A B . . . . −0.54 0.44 * . . −0.60 0.26 Arg 151 . A B . . . . −0.79 0.51 * * . −0.60 0.26 Gly 152 . A B . . . . −1.39 0.59 * * . −0.60 0.32 Leu 153 A A . . . . . −1.04 0.59 * * . −0.60 0.31 Ala 154 A A . . . . . −0.48 0.20 * * . −0.30 0.27 Ala 155 A A . . . . . −0.59 0.96 * * . −0.60 0.43 Leu 156 A A . . . . . −0.94 1.21 . * . −0.60 0.43 Gln 157 . A B . . . . −1.41 1.29 . . . −0.60 0.67 Tyr 158 . A B . . . . −0.60 1.47 . . . −0.60 0.55 Leu 159 . A B . . . . −0.01 1.37 . . . −0.45 1.15 Tyr 160 . A B . . . . 0.58 0.69 . . . −0.45 1.11 Leu 161 . . B . . T . 0.80 0.69 . . . −0.05 1.14 Gln 162 . . B . . T . −0.01 0.43 . . . −0.05 1.39 Asp 163 . . B . . T . 0.23 0.43 . . . −0.20 0.73 Asn 164 . . B . . T . 0.46 0.07 * . . 0.25 1.54 Ala 165 . A B . . . . −0.11 −0.11 * . . 0.30 0.90 Leu 166 . A B . . . . 0.49 0.17 * . . −0.30 0.44 Gln 167 . A B . . . . 0.49 0.60 * . . −0.32 0.43 Ala 168 . A B . . . . 0.49 0.20 * . . 0.26 0.70 Leu 169 . . B . . T . 0.18 −0.30 * . . 1.69 1.43 Pro 170 . . B . . T . 0.07 −0.50 * * F 2.12 1.19 Asp 171 . . . . T T . 0.99 −0.11 * * F 2.80 1.02 Asp 172 . . B . . T . 0.99 −0.61 * . F 2.42 2.42 Thr 173 . . B . . . . 0.77 −1.30 * . F 1.94 2.61 Phe 174 . . B . . . . 1.23 −1.04 * . F 1.87 1.29 Arg 175 . . B . . . . 1.44 −0.61 * * F 1.65 0.76 Asp 176 A . . . . . . 0.63 −0.21 * * F 1.28 0.85 Leu 177 . . . . T . . 0.32 −0.01 * * F 1.89 0.81 Gly 178 . . . . T . . 0.60 −0.31 * * F 2.10 0.60 Asn 179 . . . . . . C 0.49 0.19 * * . 0.94 0.49 Leu 180 A . . B . . . −0.32 0.87 * . . 0.03 0.49 Thr 181 . . B B . . . −1.13 0.97 * . . −0.18 0.43 His 182 . . B B . . . −0.36 1.23 * . . −0.39 0.22 Leu 183 . . B B . . . −0.36 1.33 . * . −0.60 0.36 Phe 184 . . B B . . . −0.36 1.07 * . . −0.60 0.25 Leu 185 . . B B . . . 0.57 0.99 * . . −0.60 0.29 His 186 . . B . . T . −0.01 0.49 * . . −0.20 0.69 Gly 187 . . . . T T . −0.28 0.49 * . F 0.35 0.56 Asn 188 . . . . T T . 0.23 0.09 * . F 0.65 0.91 Arg 189 . . . . T T . 0.08 −0.21 . . F 1.25 0.90 Ile 190 . . . . . . C 0.68 −0.07 . . F 0.85 0.67 Ser 191 . . . . . . C 0.71 −0.07 * . F 0.85 0.65 Ser 192 . . B . . . . 1.17 −0.47 * * F 0.65 0.57 Val 193 . . B . . . . 0.58 −0.47 * . F 0.80 1.60 Pro 194 . A B . . . . −0.23 −0.66 * . F 0.90 1.21 Glu 195 . A B . . . . 0.77 −0.26 * . F 0.45 0.78 Arg 196 A A . . . . . 0.72 −0.64 * . F 0.90 2.06 Ala 197 A A . . . . . 0.21 −0.86 * . . 0.75 1.32 Phe 198 A A . . . . . 1.03 −0.60 * . . 0.60 0.63 Arg 199 A A . . . . . 0.94 −0.10 * . . 0.30 0.44 Gly 200 A A . . . . . 0.13 0.29 * * . −0.30 0.58 Leu 201 A A . . . . . 0.02 0.47 * . . −0.60 0.55 His 202 . A . . . . C 0.72 −0.31 * * . 0.50 0.47 Ser 203 . A . . . . C 0.61 −0.31 * * . 0.50 0.93 Leu 204 A A . . . . . −0.31 −0.06 * * . 0.30 0.93 Asp 205 A A . . . . . −0.78 −0.06 * . . 0.30 0.56 Arg 206 A A . B . . . 0.00 0.13 * . . −0.30 0.35 Leu 207 A A . B . . . 0.03 0.24 * . . −0.30 0.57 Leu 208 A A . B . . . 0.33 −0.04 * . . 0.30 0.59 Leu 209 A A . B . . . 1.26 0.36 * . . −0.30 0.49 His 210 A . . . . T . 0.40 0.36 * . . 0.25 1.16 Gln 211 A . . . . T . −0.30 0.31 * * F 0.40 1.04 Asn 212 A . . . . T . 0.48 0.13 . . F 0.40 1.28 Arg 213 A . . . . T . 0.43 −0.06 . . . 0.85 1.28 Val 214 A A . . . . . 1.21 0.09 . . . −0.30 0.55 Ala 215 . A B . . . . 1.03 0.19 . . . −0.30 0.46 His 216 . A B . . . . 1.00 0.21 . * . −0.30 0.37 Val 217 . A B . . . . 0.41 0.71 * . . −0.60 0.67 His 218 . A B . . . . −0.40 0.57 * . . −0.60 0.67 Pro 219 . A B . . . . 0.57 0.86 * . . −0.60 0.43 His 220 A . . . . . . 1.16 0.36 * . . 0.05 1.12 Ala 221 A . . . . . . 0.38 −0.29 * . . 0.65 1.38 Phe 222 A . . . . . . 0.89 −0.10 * * . 0.50 0.74 Arg 223 A . . . . . . 1.03 −0.10 * * . 0.50 0.54 Asp 224 A . . . . . . 0.43 −0.60 * * F 1.10 1.04 Leu 225 A . . . . . . −0.13 −0.41 * * F 0.65 0.99 Gly 226 A . . . . . . 0.14 −0.59 * * F 0.95 0.50 Arg 227 A . . B . . . 0.03 −0.10 * * . 0.30 0.43 Leu 228 . . B B . . . −0.32 0.59 * * . −0.60 0.43 Met 229 . . B B . . . −1.13 0.66 * . . −0.60 0.68 Thr 230 . . B B . . . −1.02 0.91 . . . −0.60 0.29 Leu 231 . . B B . . . −1.27 1.70 . * . −0.60 0.30 Tyr 232 . . B B . . . −1.38 1.51 . . . −0.60 0.31 Leu 233 . . B B . . . −0.57 1.30 . . . −0.60 0.34 Phe 234 A . . . . T . −0.78 1.21 . . . −0.20 0.67 Ala 235 A . . . . T . −0.77 1.21 . . . −0.20 0.35 Asn 236 A . . . . T . −0.54 0.84 * . . −0.20 0.57 Asn 237 . . . . . T C −1.11 0.66 . . . 0.00 0.67 Leu 238 . A . . . . C −0.51 0.56 . . . −0.40 0.55 Ser 239 . A . . . . C −0.12 0.49 * . . −0.40 0.52 Ala 240 . A . . . . C 0.47 0.57 * . . −0.40 0.47 Leu 241 . A . . . . C −0.12 0.17 * . . −0.10 0.99 Pro 242 A A . . . . . −0.93 −0.01 . . F 0.45 0.75 Thr 243 A A . . . . . −0.71 0.29 . . F −0.15 0.61 Glu 244 A A . . . . . −0.62 0.29 . . F −0.15 0.75 Ala 245 A A . . . . . −0.84 0.03 * * . −0.30 0.75 Leu 246 A A . . . . . 0.08 0.29 * * . −0.30 0.43 Ala 247 A A . . . . . −0.30 −0.20 * * . 0.30 0.48 Pro 248 A A . . . . . −0.80 0.30 * * . −0.30 0.48 Leu 249 A A . . . . . −0.80 0.49 * * . −0.60 0.48 Arg 250 A A . . . . . −0.46 0.20 * * . −0.30 0.83 Ala 251 A A . . . . . −0.46 0.46 * * . −0.60 0.84 Leu 252 A A . . . . . 0.24 0.71 * * . −0.60 0.84 Gln 253 . A B . . . . −0.36 0.03 * * . −0.30 0.84 Tyr 254 . A B . . . . 0.46 0.71 . * . −0.60 0.68 Leu 255 . A B . . . . 0.34 0.61 * * . −0.17 1.33 Arg 256 . A B . . . . 0.93 −0.07 * * . 1.01 1.28 Leu 257 . A . . T . . 1.53 −0.07 * * . 1.69 1.32 Asn 258 . . . . T T . 1.24 −0.40 * * F 2.52 2.47 Asp 259 . . . . T T . 0.63 −0.17 . * F 2.80 1.33 Asn 260 . . . . . T C 0.78 0.47 . * F 1.42 1.19 Pro 261 . . . . T T . 0.67 0.36 . * F 1.49 0.40 Trp 262 . . . . T . . 0.81 −0.04 . * . 1.46 0.40 Val 263 . . B . . . . 0.92 0.53 . * . 0.19 0.13 Cys 264 . . B . . T . 0.33 0.13 . * . 0.72 0.17 Asp 265 . . B . . T . 0.44 0.20 . * . 1.03 0.16 Cys 266 . . B . . T . 0.44 −0.71 . * . 2.24 0.43 Arg 267 . . . . T T . −0.08 −0.93 . * . 3.10 1.23 Ala 268 . . . . T . . 0.49 −0.81 . * . 2.44 0.61 Arg 269 . . . . . T C 0.57 0.10 . * . 1.38 1.19 Pro 270 . . . . . T C 0.28 0.03 . * . 0.92 0.61 Leu 271 A . . . . T . 0.13 0.94 . * . 0.11 0.64 Trp 272 A . . . . T . 0.02 1.13 * * . −0.20 0.27 Ala 273 A A . . . . . 0.66 1.53 * . . −0.60 0.30 Trp 274 A A . . . . . −0.16 1.10 * * . −0.60 0.73 Leu 275 A A . . . . . 0.17 1.20 * * . −0.60 0.60 Gln 276 . A B . . . . 0.63 0.29 * * . −0.15 1.17 Lys 277 . A B . . . . 0.62 0.21 . * F 0.34 1.10 Phe 278 . A . . T . . 0.91 −0.31 . * F 1.68 1.79 Arg 279 . A . . T . . 0.90 −0.61 * * F 2.32 1.38 Gly 280 . . . . T T . 1.71 −0.63 * * F 2.91 0.93 Ser 281 . . . . T T . 0.86 −0.63 * * F 3.40 1.85 Ser 282 . . . . . T C 0.60 −0.77 . * F 2.71 0.70 Ser 283 . . . . T T . 0.63 −0.34 . * F 2.42 1.10 Glu 284 . . . . T . . 0.22 −0.20 . * F 1.73 0.44 Val 285 . . B . . T . −0.24 −0.20 . . F 1.19 0.44 Pro 286 . . . . T T . −0.16 0.10 . . . 0.50 0.27 Cys 287 . . . . T T . 0.14 0.14 . * . 0.50 0.24 Ser 288 . . B . . T . 0.56 0.54 * * . −0.20 0.56 Leu 289 . . B . . . . −0.26 −0.10 * * F 0.65 0.71 Pro 290 . . B . . . . 0.01 0.16 . * F 0.20 1.10 Gln 291 . A B . . . . −0.12 0.09 * * F −0.15 0.83 Arg 292 . A B . . . . 0.66 0.13 * * F −0.15 0.99 Leu 293 . A B . . . . 0.96 −0.56 * * F 0.90 1.26 Ala 294 . A B . . . . 0.96 −0.99 * * F 0.90 1.21 Gly 295 A . . . . T . 1.21 −0.70 * * F 1.15 0.51 Arg 296 A . . . . T . 1.32 −0.70 * * F 1.30 1.24 Asp 297 A . . . . T . 0.40 −1.39 * * F 1.30 2.40 Leu 298 A . . . . T . 0.62 −1.20 * * F 1.30 2.00 Lys 299 A A . . . . . 0.62 −1.13 * * F 0.90 1.03 Arg 300 A A . . . . . 0.97 −0.63 * . . 0.60 0.62 Leu 301 A A . . . . . 0.86 −0.23 * . . 0.45 1.22 Ala 302 A A . . . . . 0.04 −0.91 * . . 0.75 1.01 Ala 303 A A . . . . . 0.86 −0.23 * . . 0.30 0.43 Asn 304 A A . . . . . 0.47 0.17 * . . −0.30 0.90 Asp 305 A A . . . . . −0.31 −0.09 * * F 0.45 0.88 Leu 306 A . . . . T . −0.09 −0.01 . . F 0.85 0.47 Gln 307 . . B . . T . −0.36 −0.01 . . . 0.70 0.29 Gly 308 . . B . . T . −0.36 0.23 . . . 0.10 0.13 Cys 309 . . B . . T . −0.67 0.73 . . . −0.20 0.16 Ala 310 . . B B . . . −1.01 0.53 . * . −0.60 0.13 Val 311 . . B B . . . −0.41 0.56 . . . −0.60 0.13 Ala 312 . . B B . . . −0.66 0.56 . . . −0.60 0.38 Thr 313 . . B B . . . −0.34 0.74 . . F −0.45 0.59 Gly 314 . . B . . T . 0.11 0.74 . . F 0.10 1.09 Pro 315 . . . . T T . −0.19 0.53 . . F 0.50 1.67 Tyr 316 . . . . . T C 0.38 0.71 . . . 0.00 0.81 His 317 . . B . . T . 0.66 1.14 . . . −0.20 0.86 Pro 318 . . B . . . . 0.62 1.20 . * . −0.40 0.80 Ile 319 . . B . . . . 1.08 1.20 . * . −0.40 0.51 Trp 320 . . B . . T . 0.70 0.44 . * . −0.20 0.73 Thr 321 . . B . . T . 0.63 0.44 . * F −0.05 0.48 Gly 322 . . . . . T C 0.67 0.50 . * F 0.15 0.98 Arg 323 . . . . . T C 0.88 −0.19 . * F 1.20 1.56 Ala 324 . A . . . . C 1.77 −1.10 . . F 1.10 1.87 Thr 325 . A . . . . C 1.84 −1.59 . * F 1.41 3.28 Asp 326 . A . . . . C 1.34 −1.59 . * F 1.72 2.59 Glu 327 . A B . . . . 1.34 −0.90 . * F 1.83 2.11 Glu 328 A . . . . T . 0.42 −0.97 . * F 2.54 1.45 Pro 329 . . . . T T . 0.80 −0.77 . . F 3.10 0.72 Leu 330 . . . . T T . 1.16 −0.34 . . F 2.49 0.64 Gly 331 . . . . T T . 0.49 −0.34 . . F 2.18 0.74 Leu 332 . . . . . . C −0.18 0.23 . . . 0.72 0.26 Pro 333 . . . . T T . −0.18 0.37 . . . 0.81 0.17 Lys 334 . . . . T T . −0.18 0.09 . . . 0.50 0.29 Cys 335 . . B . . T . 0.63 0.09 . . . 0.10 0.55 Cys 336 . . B . . T . 0.39 −0.60 . . . 1.00 0.59 Gln 337 . . B . . T . 0.61 −0.53 . . . 1.00 0.30 Pro 338 . . B . . T . 0.82 −0.03 . . F 0.85 0.56 Asp 339 A . . . . T . 0.82 −0.60 . . F 1.30 1.75 Ala 340 A . . . . T . 0.90 −1.17 . * F 1.30 2.02 Ala 341 A A . . . . . 1.27 −1.07 . * F 0.90 1.32 Asp 342 A A . . . . . 0.41 −1.11 . * F 0.90 1.06 Lys 343 A A . . . . . −0.19 −0.47 . . F 0.45 0.78 Ala 344 A A . . . . . −0.19 −0.29 . . F 0.45 0.63 Ser 345 . A B . . . . 0.19 −0.79 . . . 0.60 0.66 Val 346 . A B . . . . 0.43 −0.36 . * . 0.64 0.51 Leu 347 . A B . . . . 0.54 0.07 . * . 0.38 0.50 Glu 348 . . B . . T . 0.29 −0.43 * * F 1.87 0.73 Pro 349 . . . . T T . 0.29 −0.39 * . F 2.76 1.52 Gly 350 . . . . T T . 0.29 −0.53 * . F 3.40 1.86 Arg 351 . . . . . T C 0.56 −0.83 * . F 2.86 1.44 Pro 352 . . . . . . C 1.02 −0.33 . . F 1.87 0.94 Ala 353 A . . . . . . 1.02 −0.33 . . F 1.33 0.94 Ser 354 A . . . . T . 0.64 −0.36 . . F 1.19 0.77 Ala 355 A . . . . T . 0.18 0.14 . * F 0.25 0.50 Gly 356 A . . . . T . 0.11 0.40 . * F 0.25 0.41 Asn 357 . . B . . T . −0.02 −0.10 . * . 0.70 0.61 Ala 358 . . B . . . . 0.68 −0.06 . * F 0.65 0.60 Leu 359 . . B . . . . 0.12 −0.56 . * F 1.10 1.19 Lys 360 . . B . . . . 0.50 −0.34 . * F 0.65 0.55 Gly 361 . . B . . . . 0.63 −0.31 . * F 0.96 0.84 Arg 362 . . B . . . . 0.29 −0.39 . * F 1.42 1.58 Val 363 . . B . . . . 0.88 −0.64 . * F 1.88 0.78 Pro 364 . . B . . T . 1.39 −0.64 . * F 2.54 1.32 Pro 365 . . . . T T . 1.13 −0.69 . * F 3.10 0.90 Gly 366 . . . . T T . 1.27 −0.26 . * F 2.64 1.88 Asp 367 . . . . T T . 0.81 −0.47 . . F 2.33 1.88 Ser 368 . . . . . . C 1.67 −0.47 . . F 1.62 1.20 Pro 369 . . . . . T C 1.53 −0.50 . . F 1.81 1.95 Pro 370 . . . . T T . 1.44 −0.50 . . F 2.00 1.16 Gly 371 . . . . T T . 1.44 −0.11 . . F 2.00 1.16 Asn 372 . . . . T T . 1.23 −0.07 . . F 2.15 0.74 Gly 373 . . . . T T . 1.64 −0.07 . . F 2.45 0.74 Ser 374 . . . . . T C 1.82 −0.50 * . F 3.00 1.47 Gly 375 . . . . . T C 1.14 −0.43 * . F 2.40 1.24 Pro 376 . . B . . T . 1.49 −0.14 * . F 1.75 0.88 Arg 377 . . B . . . . 1.49 −0.17 * . F 1.40 1.05 His 378 . . B . . . . 1.53 −0.56 * . . 1.25 1.78 Ile 379 . . B . . . . 1.62 −0.60 * . . 1.20 1.54 Asn 380 . . B . . . . 1.27 −0.60 * . F 1.60 1.22 Asp 381 . . . . T . . 1.13 0.19 * . F 1.20 0.77 Ser 382 . . . . . T C 0.71 0.11 * . F 1.60 1.09 Pro 383 . . . . T T . −0.07 −0.09 . . F 2.50 0.98 Phe 384 . . . . T T . 0.61 0.20 . . F 1.65 0.48 Gly 385 . . . . T T . 0.27 0.63 . . F 1.10 0.56 Thr 386 . . B . . . . −0.03 0.67 . . F 0.49 0.36 Leu 387 . . . . . T C −0.32 0.63 . . F 0.88 0.55 Pro 388 . . . . . T C −0.11 0.34 . * F 1.17 0.57 Gly 389 . . . . T T . 0.38 −0.09 . . F 2.21 0.68 Ser 390 . . . . . T C 0.51 −0.14 . * F 2.40 1.27 Ala 391 . . . . . . C 0.23 −0.40 . * F 1.96 1.27 Glu 392 . . . . . . C 1.01 −0.33 . * F 1.72 1.30 Pro 393 . . B . . . . 0.56 −0.26 . * F 1.28 1.32 Pro 394 A . . . . T . 0.60 −0.07 . * F 1.09 0.70 Ala 395 A . . . . T . 0.31 −0.19 . * . 0.70 0.54 His 396 A . . . . T . 0.31 0.31 . * . 0.10 0.35 Cys 397 A . . . . T . 0.42 0.39 . * . 0.10 0.23 Ser 398 A . . . . . . 0.29 −0.04 * . . 0.50 0.45 Ala 399 A . . . . . . −0.31 −0.11 * . . 0.50 0.33 Ala 400 A . . . . . . 0.39 0.07 * . . −0.10 0.50 Arg 401 A . . . . . . −0.17 −0.50 * . . 0.80 0.73 Gly 402 A . . . . . . 0.19 −0.39 * . . 0.50 0.73 Leu 403 . . B B . . . 0.60 −0.40 * . . 0.45 1.05 Arg 404 . . B B . . . 0.49 −0.90 * . . 0.75 1.05 Ala 405 . . B B . . . 0.87 −0.11 * . . 0.30 0.92 Thr 406 . . B B . . . 0.44 −0.11 * . F 0.60 1.72 Arg 407 . . B B . . . 0.49 −0.31 * . F 0.60 1.26 Phe 408 . . B . . T . 0.96 0.07 * * F 0.40 1.68 Pro 409 . . . . T T . 0.63 0.00 * . F 1.74 1.15 Thr 410 . . . . T T . 1.33 −0.06 * * F 1.93 0.91 Ser 411 . . . . . T C 1.76 −0.06 * * F 2.22 2.05 Gly 412 . . . . . T C 1.76 −0.84 * * F 2.86 2.60 Pro 413 . . . . T T . 2.24 −1.27 . . F 3.40 3.53 Arg 414 . . . . T T . 2.11 −1.33 . . F 3.06 4.08 Arg 415 . . . . T T . 1.76 −1.29 . . F 2.72 4.08 Arg 416 . . B . . T . 1.76 −1.14 . . F 1.98 1.41 Pro 417 . . . . T T . 2.21 −1.19 . . F 2.23 0.97 Gly 418 . . . . T T . 2.47 −1.19 . . F 2.23 0.97 Cys 419 . . . . T T . 2.36 −1.19 . . F 2.57 0.99 Ser 420 . . . . T . . 2.36 −0.79 . . F 2.86 1.03 Arg 421 . . . . T T . 1.93 −1.21 . * F 3.40 2.03 Lys 422 . . . . T T . 2.26 −1.16 . * F 3.06 5.47 Asn 423 . . . . T T . 2.30 −1.73 . * F 2.98 7.99 Arg 424 . . . . T T . 2.93 −1.73 . * F 2.90 5.47 Thr 425 . . . . T . . 2.57 −1.23 . * F 2.62 3.72 Arg 426 . . . . T T . 2.57 −0.66 . * F 2.74 1.24 Ser 427 . . B . . T . 1.71 −1.06 * * F 2.60 1.24 His 428 . . B . . T . 1.37 −0.37 * * . 1.74 0.71 Cys 429 . . B . . T . 1.26 −0.43 * * . 1.48 0.36 Arg 430 . . B . . . . 0.98 −0.03 * * . 1.02 0.46 Leu 431 . . B . . . . 0.52 0.09 * * . 0.16 0.34 Gly 432 . . B . . . . 0.52 0.01 . * . −0.10 0.63 Gln 433 . . B . . . . 0.21 −0.17 * * F 0.65 0.43 Ala 434 . . B . . . . 0.53 0.26 * . F 0.05 0.52 Gly 435 . . . . . T C 0.08 0.00 * . F 1.05 0.52 Ser 436 . . . . . T C 0.54 0.00 . . F 1.05 0.30 Gly 437 . . . . . T C 0.58 0.03 . . F 0.45 0.29 Gly 438 . . . . . T C 0.23 0.01 . . F 0.71 0.43 Gly 439 . . . . . T C 0.82 0.01 . . F 0.97 0.31 Gly 440 . . . . . T C 0.87 −0.37 . . F 1.83 0.53 Thr 441 . . . . . T C 1.17 −0.41 . . F 2.09 0.72 Gly 442 . . B . . T . 1.17 −0.84 . . F 2.60 1.26 Asp 443 . . B . . T . 1.21 −0.84 . * F 2.34 1.26 Ser 444 . . B . . T . 1.21 −0.89 . . F 2.29 1.17 Glu 445 . . B . . T . 0.97 −0.94 . * F 2.24 1.17 Gly 446 . . . . T T . 0.47 −0.87 . * F 2.44 0.71 Ser 447 . . . . T . . 0.60 −0.19 . * F 1.89 0.43 Gly 448 . . . . T . . 0.30 −0.14 . . F 2.10 0.39 Ala 449 . . . . . . C −0.21 0.24 . . F 1.09 0.52 Leu 450 . . B . . T . −0.52 0.50 . . F 0.58 0.32 Pro 451 . . B . . T . −0.84 0.60 . . F 0.37 0.47 Ser 452 . . B . . T . −0.84 0.74 . . F 0.16 0.25 Leu 453 . . B . . T . −1.31 0.63 . . . −0.20 0.41 Thr 454 . . B . . . . −1.03 0.63 . . . −0.40 0.22 Cys 455 . . B . . . . −0.43 0.69 * . . −0.40 0.23 Ser 456 . . B . . . . −1.03 0.73 . . . −0.40 0.44 Leu 457 . . B . . . . −1.08 0.73 . . . −0.40 0.25 Thr 458 . . B . . T . −1.08 0.67 . . F −0.05 0.46 Pro 459 . . B . . T . −1.36 0.79 . . F −0.05 0.28 Leu 460 . . B . . T . −1.50 0.90 . . . −0.20 0.35 Gly 461 . . B . . T . −2.06 0.90 . . . −0.20 0.20 Leu 462 . . B B . . . −2.06 1.06 . . . −0.60 0.10 Ala 463 . . B B . . . −2.03 1.31 . . . −0.60 0.10 Leu 464 . . B B . . . −2.13 1.54 . . . −0.60 0.10 Val 465 . . B B . . . −2.18 1.60 . . . −0.60 0.18 Leu 466 . . B B . . . −2.64 1.56 . . . −0.60 0.13 Trp 467 . . B B . . . −2.18 1.74 . . . −0.60 0.13 Thr 468 . . B B . . . −1.80 1.49 . . . −0.60 0.17 Val 469 . . B B . . . −1.66 1.27 . . . −0.60 0.33 Leu 470 . . B B . . . −1.19 1.16 . . . −0.60 0.17 Gly 471 . . . B . . C −0.77 0.67 . . . −0.40 0.15 Pro 472 . . . . T . . −0.87 0.61 . . . 0.00 0.25 Cys 473 . . . . T . . −0.94 0.40 . . . 0.30 0.39

TABLE 10 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . . B . . . . 0.59 −0.19 . * . 0.86 1.62 Arg 2 . . B . . . . 0.77 −0.19 . * . 1.07 1.25 Thr 3 . . . . . T C 0.34 −0.19 . * . 1.68 1.52 Pro 4 . . . . . T C 0.52 0.07 . * . 1.29 1.26 Gly 5 . . . . . T C 0.06 −0.11 . * F 2.10 1.00 Pro 6 . . . . . T C −0.16 0.53 . * F 0.99 0.51 Leu 7 . A B . . . . −1.08 0.73 . * F 0.18 0.27 Pro 8 . A B . . . . −1.58 0.99 . . . −0.18 0.23 Val 9 . A B . . . . −2.18 1.24 . . . −0.39 0.12 Leu 10 . A B . . . . −2.64 1.50 . . . −0.60 0.12 Leu 11 . A B . . . . −3.02 1.50 . . . −0.60 0.06 Leu 12 . A B . . . . −2.56 1.57 . . . −0.60 0.09 Leu 13 . A B . . . . −2.93 1.36 . . . −0.60 0.11 Leu 14 . A B . . . . −2.29 1.17 . . . −0.60 0.13 Ala 15 . A B . . . . −2.07 0.91 . . . −0.60 0.24 Gly 16 . A B . . . . −1.84 0.73 . . . −0.60 0.30 Ala 17 . . B . . . . −0.92 0.54 . . . −0.40 0.37 Pro 18 . . B . . . . −0.32 −0.14 . . . 0.74 0.71 Ala 19 . . . . T . . 0.18 −0.21 . . . 1.53 1.11 Ala 20 . . B . . . . 0.56 −0.16 . . . 1.37 1.58 Arg 21 . . B . . . . 0.69 −0.23 . . F 1.76 1.58 Pro 22 . . . . T . . 0.97 −0.23 . . F 2.40 2.42 Thr 23 . . . . . . C 0.51 −0.24 . . F 1.96 3.46 Pro 24 . . . . . T C 0.86 −0.17 . . F 1.77 0.95 Pro 25 . . . . T T . 1.14 0.59 . * F 0.83 0.96 Thr 26 . . . . T T . 1.14 0.54 . * F 0.59 0.89 Cys 27 . . B . . T . 0.76 0.06 . * . 0.25 1.13 Tyr 28 . . B . . . . 1.18 0.24 . * . −0.10 0.72 Ser 29 . A B . . . . 0.80 −0.19 . * . 0.30 0.98 Arg 30 . A B . . . . 0.20 −0.17 . * . 0.45 1.85 Met 31 . A B . . . . 0.21 −0.06 . * . 0.30 0.97 Arg 32 . A B . . . . 0.88 −0.43 . * . 0.30 0.97 Ala 33 . A B . . . . 1.12 −0.41 * * . 0.30 0.86 Leu 34 . A . . . . C 0.53 −0.41 * * . 0.65 1.50 Ser 35 . A B . . . . 0.11 −0.34 * * F 0.45 0.54 Gln 36 . A B . . . . 0.82 0.14 * . F −0.15 0.77 Glu 37 . A B . . . . 0.71 −0.36 * . F 0.60 1.83 Ile 38 . A B . . . . 0.60 −1.04 * . F 0.90 2.28 Thr 39 . A B . . . . 1.41 −0.64 * * F 0.90 1.14 Arg 40 . A B . . . . 0.90 −0.64 * . F 0.90 1.06 Asp 41 . A . . T . . 0.09 0.04 * . F 0.40 1.24 Phe 42 . . B B . . . 0.09 0.04 * . . −0.30 0.71 Asn 43 . . B B . . . 0.12 −0.04 * . . 0.30 0.63 Leu 44 . . . B . . C 0.13 0.60 . . . −0.40 0.28 Leu 45 . . B B . . . 0.02 0.99 . . . −0.60 0.43 Gln 46 . . B B . . . −0.19 0.20 . . . 0.04 0.47 Val 47 . . . B . . C 0.21 0.23 . . . 0.58 0.87 Ser 48 . . . B . . C 0.21 −0.07 . . F 1.82 1.42 Glu 49 . . . . . T C 0.81 −0.76 . . F 2.86 1.42 Pro 50 . . . . T T . 0.96 −0.73 . . F 3.40 2.95 Ser 51 . . . . T T . 0.10 −0.80 * * F 3.06 1.18 Glu 52 . . . . . T C 1.07 −0.54 * * F 2.37 0.51 Pro 53 . . . B T . . 1.12 −0.54 * . F 1.83 0.64 Cys 54 . . B B . . . 0.31 −0.21 * . . 0.64 0.75 Val 55 . . B B . . . 0.31 0.09 * * . −0.30 0.36 Arg 56 . . B B . . . 0.72 0.51 * * . −0.60 0.36 Tyr 57 . . B B . . . −0.09 0.09 * * . −0.15 1.30 Leu 58 . . B B . . . −0.12 0.20 * * . −0.15 1.45 Pro 59 . . B B . . . −0.27 0.31 * * . −0.15 1.16 Arg 60 . . B B . . . 0.59 1.00 * * . −0.60 0.61 Leu 61 . . B B . . . −0.41 0.24 * * . −0.15 1.24 Tyr 62 . . B B . . . −0.20 0.24 * * . −0.30 0.56 Leu 63 . . B B . . . 0.61 0.31 * * . −0.30 0.39 Asp 64 . . B B . . . 0.58 0.71 * * . −0.60 0.76 Ile 65 . . B B . . . −0.20 0.79 * * . −0.60 0.76 His 66 . . B . . T . −0.24 0.60 . * . −0.20 0.49 Asn 67 . . B . . T . −0.81 0.56 . * . −0.20 0.22 Tyr 68 . . B . . T . 0.00 1.24 . * . −0.20 0.26 Cys 69 . . B . . T . 0.04 0.56 . . . −0.20 0.32 Val 70 . A B B . . . 0.12 0.06 . * . −0.30 0.39 Leu 71 . A B B . . . 0.27 0.34 * * . −0.30 0.21 Asp 72 . A B B . . . 0.27 −0.41 * . F 0.45 0.76 Lys 73 . A B . . . . −0.19 −0.99 * * F 0.90 1.70 Leu 74 . A B B . . . −0.38 −0.84 * . F 0.90 1.79 Arg 75 . A B B . . . −0.11 −0.89 * . F 0.75 0.79 Asp 76 . A B B . . . 0.40 −0.39 * . . 0.30 0.40 Phe 77 . A B B . . . 0.19 0.00 * . . 0.30 0.65 Val 78 . A B B . . . −0.07 −0.26 * * . 0.30 0.52 Ala 79 . A B B . . . 0.08 0.17 * . . −0.30 0.48 Ser 80 . A . B . . C −0.32 0.74 . . . −0.40 0.30 Pro 81 . . . . . T C −0.28 0.87 . * F 0.15 0.42 Pro 82 . . . . T T . −0.43 0.23 . . F 0.65 0.83 Cys 83 . . . . T T . −0.17 0.37 . . . 0.50 0.46 Trp 84 . . . . T T . 0.42 0.49 . . . 0.20 0.30 Lys 85 . A B . . . . −0.13 0.46 . . . −0.60 0.34 Val 86 . A B . . . . 0.08 0.67 . . . −0.60 0.46 Ala 87 . A B . . . . −0.01 0.10 . . . −0.30 0.74 Gln 88 . A B . . . . −0.16 −0.43 . . . 0.30 0.49 Val 89 . A B . . . . 0.18 0.26 . . . −0.30 0.55 Asp 90 . A B . . . . 0.13 −0.39 . . F 0.60 1.09 Ser 91 . A B . . . . 1.03 −0.89 . . F 0.90 1.05 Leu 92 A A . . . . . 1.03 −1.29 * * F 0.90 2.83 Lys 93 A A . . . . . 1.14 −1.43 * * F 0.90 1.71 Asp 94 . A . . T . . 2.04 −1.43 * . F 1.30 2.50 Lys 95 A A . . . . . 1.23 −1.81 * * F 0.90 6.06 Ala 96 A A . . . . . 1.29 −1.81 * * F 0.90 2.50 Arg 97 . A B . . . . 1.79 −1.06 * * F 0.90 2.34 Lys 98 . A B . . . . 0.86 −0.57 * * F 0.90 1.69 Leu 99 . A B . . . . 0.26 0.11 * . . −0.15 1.17 Tyr 100 . A B . . . . 0.21 0.23 * . . −0.30 0.59 Thr 101 . . B B . . . 0.50 0.63 * . . −0.60 0.48 Ile 102 . . B B . . . −0.31 1.01 * . . −0.60 0.77 Met 103 . . B B . . . −1.02 1.11 * . . −0.60 0.43 Asn 104 . . B . . T . −0.10 0.93 * . . 0.04 0.16 Ser 105 . . B . . T . 0.26 0.44 * . . 0.28 0.44 Phe 106 . . B . . T . 0.57 −0.24 * . . 1.42 0.88 Cys 107 . . B . . T . 0.64 −0.86 . . . 1.96 0.91 Arg 108 . . . . T . . 0.39 −0.57 . . . 2.40 0.56 Arg 109 . . B B . . . −0.31 −0.31 * . F 1.41 0.48 Asp 110 . . B B . . . −0.82 −0.31 . . F 1.17 0.78 Leu 111 . . B B . . . −0.93 −0.20 * . . 0.78 0.33 Val 112 . . B B . . . −0.27 0.49 * . . −0.36 0.14 Phe 113 . . B B . . . −0.38 0.49 * . . −0.60 0.14 Leu 114 . . B B . . . −1.16 0.49 * . . −0.60 0.28 Leu 115 . . B B . . . −1.16 0.37 . . . −0.02 0.20 Asp 116 . . . . T T . −0.93 0.13 . . F 1.21 0.37 Asp 117 . . . . T T . −0.89 −0.16 . . F 2.09 0.46 Cys 118 . . . . T T . −0.19 −0.16 . . . 2.22 0.46 Asn 119 . . . . T T . 0.38 −0.84 . . . 2.80 0.48 Ala 120 . A B . . . . 0.98 −0.09 . . . 1.42 0.45 Leu 121 . A B . . . . 0.09 0.34 . . . 0.69 1.29 Glu 122 . A B . . . . −0.12 0.46 . * . −0.04 0.56 Tyr 123 . A B . . . . −0.31 0.49 . * . −0.32 0.86 Pro 124 . . B B . . . −0.62 0.63 . * . −0.60 0.77 Ile 125 . . B B . . . −0.34 0.43 . * . −0.60 0.64 Pro 126 . . B B . . . −0.39 0.91 . * . −0.60 0.59 Val 127 . . B B . . . −1.20 0.80 . . . −0.60 0.29 Thr 128 . . B B . . . −1.17 1.06 . . . −0.60 0.34 Thr 129 . . B B . . . −0.96 0.80 . . F −0.11 0.34 Val 130 . . B B . . . 0.04 0.37 . . F 0.53 0.75 Leu 131 . . B . . T . 0.26 −0.27 . * F 2.02 1.02 Pro 132 . . B . . T . 1.22 −0.36 . * F 2.36 1.23 Asp 133 . . . . T T . 1.14 −0.84 . * F 3.40 3.24 Arg 134 . . B . . T . 1.07 −1.06 . * . 2.51 5.03 Gln 135 . . B . . . . 1.53 −1.31 . * . 1.97 4.16 Arg 136 . . B . . . . 1.96 −1.31 . * . 1.63 3.18

TABLE 11 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 A A . . . . . −0.68 0.09 . . . −0.30 0.62 Lys 2 A A . . . . . −1.10 0.23 . . . −0.30 0.26 Ala 3 A A . . . . . −1.52 0.49 * . . −0.60 0.17 Leu 4 A A . . . . . −1.94 0.74 . . . −0.60 0.14 Cys 5 A A . . . . . −2.37 0.81 . . . −0.60 0.06 Leu 6 A A . . . . . −1.98 1.50 * . . −0.60 0.05 Leu 7 . A B . . . . −2.88 1.43 * . . −0.60 0.09 Leu 8 . A B . . . . −3.10 1.39 . . . −0.60 0.12 Leu 9 . A B . . . . −2.63 1.50 . . . −0.60 0.12 Pro 10 . A B . . . . −2.78 1.24 . . . −0.60 0.15 Val 11 . A B . . . . −2.78 1.24 . . . −0.60 0.15 Leu 12 . A B . . . . −2.82 1.24 . . . −0.60 0.15 Gly 13 . A B . . . . −2.31 1.20 * . . −0.60 0.07 Leu 14 . A B . . . . −1.80 1.16 . . . −0.60 0.13 Leu 15 . A B . . . . −1.54 0.90 . . . −0.60 0.21 Val 16 . . B . . T . −1.00 0.21 . . . 0.10 0.42 Ser 17 . . B . . T . −1.00 0.27 . . F 0.25 0.73 Ser 18 A . . . . T . −1.32 0.27 . . F 0.25 0.73 Lys 19 A . . . . T . −0.81 0.16 . . F 0.25 0.53 Thr 20 A . . B . . . −0.60 −0.10 . . F 0.45 0.53 Leu 21 A . . B . . . 0.26 0.13 * . . −0.30 0.39 Cys 22 A . . B . . . 0.56 −0.26 * . . 0.30 0.34 Ser 23 A . . B . . . 0.27 −0.26 * . . 0.30 0.40 Met 24 A A . . . . . −0.67 −0.24 * . . 0.30 0.49 Glu 25 A A . . . . . −0.36 −0.24 * . . 0.30 0.65 Glu 26 A A . . . . . 0.46 −0.41 * * . 0.30 0.77 Ala 27 A A . . . . . 1.23 −0.80 * * . 0.75 1.36 Ile 28 A A . . . . . 0.64 −1.41 * * . 0.75 1.53 Asn 29 A A . . . . . 1.24 −0.73 * * . 0.60 0.62 Glu 30 A A . . . . . 1.24 −0.33 * * F 0.60 1.06 Arg 31 A A . . . . . 0.39 −0.83 * * F 0.90 2.63 Ile 32 A A . . . . . 0.39 −0.87 * * F 0.90 1.21 Gln 33 A A . . . . . 0.93 −0.77 * * F 0.75 0.71 Glu 34 A A . . . . . 0.63 −0.34 * * . 0.30 0.36 Val 35 A A . . . . . −0.18 0.04 * . . −0.30 0.68 Ala 36 A A . . . . . −1.18 0.04 * * . −0.30 0.33 Gly 37 A A . B . . . −0.99 0.33 * * . −0.30 0.13 Ser 38 A A . B . . . −0.88 1.11 * * . −0.60 0.15 Leu 39 A A . B . . . −1.47 0.47 * * . −0.60 0.30 Ile 40 . A B B . . . −1.50 0.47 * * . −0.60 0.30 Phe 41 . A B B . . . −1.21 0.73 * . . −0.60 0.16 Arg 42 . A B B . . . −1.17 0.73 * * . −0.60 0.26 Ala 43 . A B B . . . −1.76 0.43 * * . −0.60 0.49 Ile 44 . A B B . . . −1.29 0.43 * * . −0.60 0.40 Ser 45 . A . B . . C −1.21 0.07 * * . −0.10 0.20 Ser 46 . . . B . . C −0.51 0.76 * * . −0.40 0.17 Ile 47 . . . B T . . −1.29 0.26 * * . 0.10 0.41 Gly 48 . A B . . . . −0.70 0.14 . . . −0.30 0.16 Leu 49 . A . . . . C −0.11 0.16 * . . −0.10 0.21 Glu 50 . A B . . . . −0.67 0.16 . . . −0.30 0.40 Cys 51 . A B B . . . −0.68 0.11 . . . −0.30 0.30 Gln 52 . . B B . . . −0.09 0.17 * * . −0.30 0.53 Ser 53 . . B B . . . 0.37 −0.13 * * F 0.45 0.41 Val 54 . . B B . . . 0.83 −0.13 . * F 0.91 1.50 Thr 55 . . B B . . . 0.83 −0.27 . * F 1.07 0.85 Ser 56 . . . . T T . 0.69 −0.67 . * F 2.63 1.07 Arg 57 . . . . T T . 0.10 −0.37 . * F 2.64 1.18 Gly 58 . . . . T T . 0.09 −0.51 . * F 3.10 0.83 Asp 59 . . . . T T . 0.28 −0.51 . * F 2.79 0.89 Leu 60 . . B . . . . 0.38 −0.33 . * F 1.58 0.24 Ala 61 . . B . . . . 0.79 0.10 * * . 0.52 0.38 Thr 62 . . B . . . . 0.33 −0.33 * * . 0.81 0.45 Cys 63 . . B . . T . −0.02 0.10 * * . 0.10 0.54 Pro 64 . . . . T T . −0.61 0.20 * . F 0.65 0.46 Arg 65 . . . . T T . −0.66 0.20 * . F 0.65 0.32 Gly 66 . . . . T T . −0.38 0.36 . . . 0.50 0.45 Phe 67 . . B B . . . −0.41 0.27 * . . −0.30 0.42 Ala 68 . . B B . . . −0.41 0.27 * . . −0.30 0.21 Val 69 . . B B . . . −0.51 0.84 * . . −0.60 0.11 Thr 70 . . B B . . . −1.29 0.90 * . . −0.60 0.19 Gly 71 . . B B . . . −1.29 0.69 . . . −0.60 0.10 Cys 72 . . . . T T . −0.89 0.61 . . . 0.20 0.13 Thr 73 . . . . T T . −0.89 0.36 . . . 0.50 0.12 Cys 74 . . . . T T . −0.70 0.37 . . . 0.50 0.13 Gly 75 . . . . T T . −0.73 0.51 . . . 0.20 0.13 Ser 76 . . . . T . . −0.69 0.37 . . . 0.30 0.09 Ala 77 . . . . T . . −0.31 0.27 . . . 0.30 0.22 Cys 78 . . . . T T . 0.00 0.61 . * . 0.20 0.23 Gly 79 . . . . T T . −0.19 0.19 . * . 0.50 0.29 Ser 80 . . . . T T . 0.27 0.44 . * . 0.20 0.21 Trp 81 . . B . . T . −0.02 −0.06 . * . 0.70 0.78 Asp 82 . A B B . . . 0.57 −0.13 . * . 0.30 0.79 Val 83 A A . B . . . 0.92 −0.56 . * . 0.75 1.02 Arg 84 A A . B . . . 0.96 −0.46 . * . 0.45 1.41 Ala 85 A A . . . . . 0.59 −0.89 . * F 0.90 1.21 Glu 86 . A . . T . . 0.84 −0.31 . * F 0.85 0.88 Thr 87 . A . . T . . 0.18 −0.46 . * F 0.85 0.61 Thr 88 . A . . T . . 1.03 0.11 . * . 0.10 0.32 Cys 89 . . . . T T . 0.26 0.01 . * . 0.50 0.32 His 90 . . . . T T . 0.26 0.59 . . . 0.20 0.12 Cys 91 . . B . . T . −0.09 0.60 . . . −0.20 0.08 Gln 92 . . B . . T . −0.38 0.54 . . . −0.20 0.16 Cys 93 . . . . T . . −0.07 0.59 . . . 0.00 0.11 Ala 94 . . . . T . . 0.31 0.09 . . . 0.30 0.35 Gly 95 . . . . T T . 0.03 0.43 . . . 0.20 0.21 Met 96 . . . . T T . 0.36 0.51 . . . 0.42 0.57 Asp 97 . . . . T T . −0.23 0.37 . . . 0.94 0.56 Trp 98 . . . . T T . 0.54 0.37 . . . 1.16 0.57 Thr 99 . . . . T . . 0.47 −0.06 . . . 1.93 1.14 Gly 100 . . . . T T . 0.14 −0.10 * . . 2.20 0.37 Ala 101 . . . . T T . 0.86 0.47 * . . 1.08 0.19 Arg 102 . . . . T T . 0.00 −0.44 * . . 1.76 0.25 Cys 103 . . . . T T . 0.29 −0.29 * . . 1.54 0.19 Cys 104 . . . . T . . 0.39 −0.31 * . . 1.12 0.32 Arg 105 . . B . . . . 0.34 −0.39 * . . 0.50 0.26 Val 106 . . B . . . . 0.54 0.04 * . . −0.10 0.61 Gln 107 . . B . . . . 0.04 −0.10 * . . 0.65 1.46 Pro 108 . . B . . . . 0.32 −0.24 . * . 0.50 0.95

TABLE 12 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . . . . . . C 0.09 0.10 . . . 0.46 0.66 Ser 2 . . . . . T C 0.48 0.16 . . . 0.84 0.74 Ser 3 . . . . . T C 0.06 −0.27 * . . 1.77 1.01 Gly 4 . . . . . T C −0.37 −0.01 . . . 1.80 0.84 Thr 5 . . . . . T C −0.27 0.06 . . F 1.17 0.52 Glu 6 A . . . . . . 0.12 0.59 * . F 0.29 0.41 Leu 7 A . . . . . . 0.08 0.63 . . . −0.04 0.63 Leu 8 A . . . . . . −0.21 0.63 . . . −0.22 0.43 Trp 9 A . . . . T . −0.46 0.64 . . . −0.20 0.25 Pro 10 A . . . . T . −0.96 1.14 . . . −0.20 0.31 Gly 11 A . . . . T . −1.77 1.14 . . . −0.20 0.31 Ala 12 A . . . . T . −1.81 1.14 . . . −0.20 0.24 Ala 13 A . . B . . . −1.81 0.87 . . . −0.60 0.12 Leu 14 A . . B . . . −2.33 1.13 . . . −0.60 0.10 Leu 15 A . . B . . . −2.47 1.39 . . . −0.60 0.08 Val 16 A . . B . . . −2.98 1.31 . . . −0.60 0.08 Leu 17 A . . B . . . −2.98 1.46 . . . −0.60 0.07 Leu 18 A . . B . . . −2.98 1.27 . . . −0.60 0.09 Gly 19 A . . B . . . −2.47 1.09 . * . −0.60 0.12 Val 20 A . . B . . . −2.47 0.83 . . . −0.60 0.19 Ala 21 A . . B . . . −2.28 0.83 . * . −0.60 0.19 Ala 22 A . . B . . . −2.32 0.71 * * . −0.60 0.10 Ser 23 A . . B . . . −1.40 0.93 * * . −0.60 0.10 Leu 24 A . . B . . . −1.72 0.29 . * . −0.30 0.20 Cys 25 . . B B . . . −1.17 0.36 * * . −0.30 0.11 Val 26 . . B B . . . −0.47 0.24 * * . −0.30 0.11 Arg 27 . . . B T . . −0.09 −0.14 * * . 1.04 0.25 Cys 28 . . . B T . . −0.13 −0.40 * * . 1.38 0.73 Ser 29 . . . B T . . 0.09 −0.54 * * F 2.17 0.97 Arg 30 . . . . . T C 0.80 −0.69 * . F 2.71 0.50 Pro 31 . . . . T T . 1.77 −0.69 * . F 3.40 1.86 Gly 32 . . . . T T . 1.36 −1.26 * . F 3.06 2.72 Ala 33 . . . . . T C 2.02 −1.26 * . F 2.52 1.86 Lys 34 . A . . . . C 2.37 −1.26 * * F 1.78 2.08 Arg 35 A A . . . . . 1.37 −1.69 * * F 1.24 4.21 Ser 36 A A . . . . . 1.33 −1.43 * . F 0.90 2.92 Glu 37 A A . . . . . 1.68 −1.17 * . F 0.90 2.29 Lys 38 A A . . . . . 2.27 −0.77 * . F 0.90 2.02 Ile 39 A A . . . . . 2.33 −0.37 * . F 0.60 2.62 Tyr 40 A . . . . . . 1.92 −0.76 . . F 1.40 2.96 Gln 41 A . . . . T . 1.41 −0.37 . * F 1.60 1.98 Gln 42 A . . . . T . 1.52 0.31 . . F 1.30 2.33 Arg 43 . . . . . T C 1.48 −0.37 . * F 2.40 2.91 Ser 44 . . . . . T C 2.37 −1.13 . . F 3.00 2.91 Leu 45 . A . . . . C 2.61 −1.53 * * F 2.30 2.81 Arg 46 . A . . T . . 2.61 −1.53 * . F 2.20 2.49 Glu 47 . A . . T . . 2.31 −1.13 * . F 1.90 3.21 Asp 48 . A . . T . . 1.50 −1.13 * . F 1.60 5.22 Gln 49 . A . . T . . 1.49 −1.03 * * F 1.30 2.31 Gln 50 . A . . T . . 1.96 −0.54 * * F 1.58 1.92 Ser 51 . A . . . . C 1.54 −0.11 * . F 1.36 1.14 Phe 52 . . . . T T . 1.66 0.27 . . F 1.49 0.88 Thr 53 . . . . T T . 1.34 −0.13 . . F 2.37 1.00 Gly 54 . . . . T T . 1.10 −0.04 * . F 2.80 1.07 Ser 55 . . . . T T . 0.80 0.33 . . F 1.92 1.94 Arg 56 . . . B T . . 0.29 −0.07 . . F 1.84 1.80 Thr 57 . . . B T . . 0.13 0.13 . . F 0.96 1.50 Tyr 58 . . . B T . . 0.10 0.34 * . . 0.38 0.83 Ser 59 . . . B . . C 0.44 0.39 * . . −0.10 0.42 Leu 60 . . B B . . . 0.16 0.79 * . . −0.60 0.50 Val 61 . . B B . . . −0.24 0.80 * . . −0.60 0.33 Gly 62 . . . . T . . −0.14 0.96 . . . 0.00 0.26 Gln 63 . . . . T . . −0.24 1.00 . . . 0.00 0.48 Ala 64 . . . . . . C −0.16 0.74 . . . −0.20 0.64 Trp 65 . . . . . T C −0.16 0.53 . . F 0.15 1.00 Pro 66 . . . . . T C 0.11 0.79 * . F 0.15 0.48 Gly 67 . . . . . T C 0.46 0.89 * . F 0.15 0.48 Pro 68 . . . . . T C −0.14 0.39 * . F 0.45 0.75 Leu 69 . A . . . . C −0.14 0.09 . . F 0.05 0.48 Ala 70 . A . . . . C −0.07 0.16 . . . −0.10 0.49 Asp 71 A A . . . . . −0.17 0.16 . . . −0.30 0.49 Met 72 A A . . . . . 0.29 0.21 . . . −0.30 0.86 Ala 73 A A . . . . . 0.54 −0.47 . . . 0.45 1.67 Pro 74 A . . . . T . 1.36 −0.97 . . F 1.30 2.00 Thr 75 A . . . . T . 1.99 −0.97 . . F 1.30 3.38 Arg 76 A . . . . T . 1.18 −1.59 . . F 1.30 6.69 Lys 77 A . . . . T . 0.97 −1.40 . . F 1.30 3.57 Asp 78 A A . . . . . 1.56 −1.14 . . F 0.90 2.04 Lys 79 A A . . . . . 1.07 −1.23 . . F 0.90 1.80 Leu 80 A A . . . . . 1.13 −0.44 . . . 0.30 0.78 Leu 81 . A B . . . . 0.81 0.31 . . . −0.30 0.73 Gln 82 . A B . . . . 0.47 0.74 * * . −0.60 0.57 Phe 83 . A B . . . . −0.34 1.13 * * . −0.60 0.92 Tyr 84 . . . . . T C −0.39 1.13 * * . 0.00 0.92 Pro 85 . . . . . T C 0.42 0.44 * * . 0.00 0.92 Ser 86 . . . . . T C 1.02 0.04 * * F 0.60 1.78 Leu 87 . . . . . T C 0.43 −0.31 * * F 1.50 1.75 Glu 88 . . . . . . C 0.83 −0.57 * * F 1.90 1.14 Asp 89 . . . . . . C 0.78 −0.61 . * F 2.20 1.14 Pro 90 . . . . . . C 1.10 −0.61 . * F 2.50 1.86 Ala 91 . . . . T . . 1.16 −1.30 . * F 3.00 2.10 Ser 92 A . . . . T . 1.97 −0.54 . * F 2.50 1.97 Ser 93 A . . . . T . 1.97 −0.14 . * F 1.90 2.21 Arg 94 A . . . . T . 1.27 −0.17 * . F 1.60 3.52 Tyr 95 . . . . T T . 1.18 0.11 * * F 1.10 2.27 Gln 96 . . . . T . . 1.81 0.11 * * F 0.94 2.27 Asn 97 . . . . T . . 1.77 −0.27 * * F 1.88 2.32 Phe 98 . . . . T . . 1.77 0.16 * * F 1.62 1.47 Ser 99 . . . . T T . 1.77 −0.21 * * F 2.76 1.13 Lys 100 . . . . T T . 1.98 −0.61 * . F 3.40 1.38 Gly 101 . . . . . T C 1.63 −0.51 * . F 2.86 2.17 Ser 102 . . . . . T C 1.33 −0.87 * . F 2.82 1.60 Arg 103 . . . . . . C 2.03 −0.87 * . F 2.58 1.07 His 104 . . . . . T C 2.33 −0.87 * . F 2.74 1.88 Gly 105 . . . . . T C 1.70 −1.30 * . F 2.70 2.43 Ser 106 . . . . . T C 1.80 −1.19 * . F 3.00 1.25 Glu 107 A . . . . T . 1.21 −0.43 . * F 2.20 1.44 Glu 108 A . . . . . . 1.10 −0.24 . * F 1.70 1.02 Ala 109 A . . . . . . 0.92 −0.67 . * . 1.55 1.27 Tyr 110 A . . . . . . 0.38 −0.63 . * . 1.25 1.14 Ile 111 A . . . . . . 0.09 0.06 . . . −0.10 0.46 Asp 112 A . . . . . . −0.51 0.56 . . . −0.40 0.46 Pro 113 A A . . . . . −0.51 0.67 . * . −0.60 0.29 Ile 114 A A . . . . . −0.17 −0.09 . . . 0.30 0.72 Ala 115 A A . . . . . −0.17 −0.01 * . . 0.30 0.67 Met 116 A A . . . . . 0.72 0.74 * . . −0.60 0.68 Glu 117 A A . . . . . 0.43 0.71 * . . −0.45 1.57 Tyr 118 A . . . . T . 0.30 0.94 * * . −0.05 1.63 Tyr 119 . . . . T T . 1.30 0.87 * * . 0.35 1.63 Asn 120 . . . . T T . 1.19 0.26 * * . 0.65 1.84 Trp 121 . . . . T T . 1.49 1.04 * * . 0.35 1.02 Gly 122 . . . . T . . 1.53 0.67 * . . 0.00 0.87 Arg 123 . . . . T . . 1.57 −0.09 * . . 1.05 1.08 Phe 124 . . . . T . . 1.60 −0.06 * . F 1.20 1.59 Ser 125 . . . . . . C 1.60 −0.54 * . F 1.64 2.49 Lys 126 . . . . . . C 1.89 −0.97 * * F 1.98 2.20 Pro 127 . . . . . T C 2.23 −0.97 * * F 2.52 4.25 Pro 128 . . . . . T C 2.12 −1.76 * * F 2.86 5.29 Glu 129 . . . . T T . 2.23 −2.14 . . F 3.40 4.42 Asp 130 A . . . . T . 2.53 −1.64 . * F 2.66 2.89 Asp 131 A . . . . . . 2.19 −1.67 . * F 2.12 3.00 Asp 132 A . . . . T . 2.16 −1.71 . . F 1.98 2.32 Ala 133 A . . . . T . 2.37 −0.96 . . F 1.64 2.18 Asn 134 A . . . . T . 2.37 −0.96 . . F 1.30 2.26 Ser 135 A . . . . T . 1.51 −0.56 * . F 1.30 2.18 Tyr 136 A . . . . . . 0.70 0.09 * . F 0.20 1.60 Glu 137 A . . . . . . −0.19 0.27 . * . −0.10 0.82 Asn 138 A . . B . . . −0.27 0.56 . . . −0.60 0.43 Val 139 A . . B . . . −0.22 0.74 . . . −0.60 0.15 Leu 140 A . . B . . . 0.08 −0.01 . . . 0.30 0.17 Ile 141 A . . B . . . 0.37 0.39 . . . −0.30 0.18 Cys 142 A . . B . . . 0.06 −0.01 . . . 0.56 0.49 Lys 143 A . . B . . . −0.26 −0.17 . . F 0.97 0.86 Gln 144 A . . . . . . 0.60 −0.37 . . F 1.58 1.77 Lys 145 . . . . . . C 1.10 −1.06 . . F 2.34 5.73 Thr 146 . . . . . . C 1.64 −1.14 . . F 2.60 4.13 Thr 147 . . . . . . C 1.72 −0.71 . * F 2.34 2.36 Glu 148 . . . . . . C 1.68 −0.61 . . F 2.08 1.19 Thr 149 . . . . . . C 1.68 −0.21 . . F 1.52 1.43 Gly 150 . A . . . . C 1.63 −0.30 . . F 1.06 1.72 Ala 151 A A . . . . . 1.60 −0.79 . . F 0.90 1.72 Gln 152 A A . . . . . 1.02 −0.36 . . F 0.60 1.18 Gln 153 A A . . . . . 0.68 −0.16 . . F 0.45 0.83 Glu 154 . A . . T . . 0.64 −0.16 . . F 0.85 0.82 Gly 155 . . . . T T . 0.18 −0.23 . . F 1.25 0.47 Ile 156 . . . . T T . 0.10 0.06 * * F 0.65 0.22 Gly 157 . . . . T T . 0.21 0.23 * * F 0.90 0.07 Gly 158 . . . . T T . −0.13 0.23 * * F 1.15 0.14 Leu 159 . . . . . . C −0.13 0.23 * * . 0.85 0.19 Cys 160 . . B . . T . −0.60 −0.46 * * . 1.70 0.32 Arg 161 . . . . T T . −0.01 −0.20 * * F 2.50 0.27 Gly 162 . . . . T T . −0.48 −0.24 . * F 2.25 0.44 Asp 163 . . . . T T . −0.43 −0.24 . * F 2.00 0.68 Leu 164 A A . . . . . −0.43 −0.43 . * F 0.95 0.46 Ser 165 A A . . . . . −0.36 0.26 . * . −0.05 0.39 Leu 166 A A . . . . . −1.28 0.33 * * . −0.30 0.23 Ser 167 A A . . . . . −0.89 1.01 . * . −0.60 0.23 Leu 168 A A . . . . . −1.20 0.33 . * . −0.30 0.35 Ala 169 A A . . . . . −0.73 0.43 . * . −0.60 0.61 Leu 170 A A . . . . . −0.64 0.17 * * . −0.05 0.45 Lys 171 . A . . T . . −0.14 0.21 * * F 0.75 0.84 Thr 172 . A . . T . . −0.14 0.01 . * F 1.15 1.20 Gly 173 . . . . . T C 0.32 −0.10 . * F 2.20 1.96 Pro 174 . . . . T T . 0.10 −0.36 . . F 2.50 0.97 Thr 175 . . . . T T . 0.24 0.33 . * F 1.65 0.55 Ser 176 . . . . T T . −0.01 0.41 . . F 1.10 0.30 Gly 177 . . . . T . . 0.00 0.41 . . F 0.65 0.30 Leu 178 . . . . . . C −0.24 0.37 . . F 0.50 0.28 Cys 179 . . . . . T C −0.33 0.39 . . F 0.75 0.21 Pro 180 . . . . . T C −0.23 0.39 . . F 1.05 0.28 Ser 181 . . . . . T C 0.07 0.39 . . F 1.35 0.53 Ala 182 . . . . . T C 0.41 −0.30 . * F 2.40 1.72 Ser 183 . . . . . T C 1.22 −0.87 . * F 3.00 1.93 Pro 184 . . . . . T C 1.89 −1.30 . . F 2.70 2.40 Glu 185 A . . . . T . 1.76 −1.69 . . F 2.20 4.12 Glu 186 A . . . . T . 1.17 −1.76 . . F 1.90 3.04 Asp 187 A . . . . T . 1.37 −1.46 . . F 1.60 1.38 Glu 188 A . . . . T . 1.28 −1.46 . . . 1.15 1.02 Gly 189 A . . . . T . 1.10 −1.03 . . . 1.00 0.75 Ile 190 A . . . . T . 0.71 −0.60 . . . 1.00 0.58

TABLE 13 Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . . B . . . . 0.43 0.01 * . . −0.10 0.79 Asp 2 . . B . . . . 0.48 −0.41 * . . 0.65 1.21 Leu 3 . . B . . T . 0.06 −0.41 * . . 0.70 0.94 Pro 4 A . . . . T . −0.41 −0.16 * . . 0.70 0.78 Arg 5 . . B . . T . −0.88 −0.13 * . F 0.85 0.35 Gly 6 . . B . . T . −0.87 0.51 * . F −0.05 0.31 Leu 7 . . B B . . . −1.16 0.33 * . . −0.30 0.20 Val 8 . . B B . . . −0.93 0.81 * . . −0.60 0.11 Val 9 . . B B . . . −1.53 1.31 . . . −0.60 0.11 Ala 10 . . B B . . . −1.94 1.57 . * . −0.60 0.11 Trp 11 . . B B . . . −2.41 1.27 . . . −0.60 0.20 Ala 12 . . B B . . . −1.89 1.31 . . . −0.60 0.22 Leu 13 . . B B . . . −1.24 1.59 . . . −0.60 0.23 Ser 14 . . . . . . C −0.73 1.51 . . . −0.20 0.34 Leu 15 . . . . . . C −0.84 1.03 . . . −0.20 0.34 Trp 16 . . . . . T C −0.87 1.31 * . . 0.00 0.35 Pro 17 . . . . . T C −0.28 1.11 * . . 0.00 0.38 Gly 18 . . . . T T . 0.22 0.73 * . F 0.35 0.77 Phe 19 . . . . T T . −0.18 0.53 * . F 0.50 1.06 Thr 20 . . . . . . C 0.63 0.40 * . F 0.25 0.59 Asp 21 . . . . . . C 0.32 0.37 . * F 0.25 0.96 Thr 22 . . . . . . C 0.53 0.56 . * . 0.29 1.10 Phe 23 . . B . . . . 0.57 −0.23 * * . 1.33 1.27 Asn 24 . . B . . T . 1.38 −0.23 * * . 1.87 1.10 Met 25 . . . . T T . 1.73 −0.23 * * . 2.61 1.49 Asp 26 . . . . T T . 1.52 −0.71 * . F 3.40 3.44 Thr 27 . . . . T T . 1.94 −1.07 * * F 3.06 3.31 Arg 28 . . . . T . . 1.79 −1.47 * * F 2.52 6.55 Lys 29 . . B . . . . 0.90 −1.44 * . F 1.78 2.91 Pro 30 . . B B . . . 1.29 −0.76 * . F 1.24 1.41 Arg 31 . . B B . . . 0.94 −0.81 . . F 0.90 1.12 Val 32 . . B B . . . 0.96 −0.39 . * . 0.30 0.55 Ile 33 . . B . . T . 0.96 0.00 . * F 0.22 0.48 Pro 34 . . B . . T . 0.60 −0.43 . * F 0.79 0.48 Gly 35 . . . . T T . 0.22 0.06 * * F 0.56 0.93 Ser 36 . . B . . T . −0.59 −0.09 * * F 0.88 1.34 Arg 37 . . B B . . . −0.43 0.01 . . F −0.30 0.75 Thr 38 . . B B . . . 0.11 0.37 . . F −0.27 0.66 Ala 39 . . B B . . . 0.08 0.37 . . . −0.39 0.49 Phe 40 . . B B . . . 0.11 0.74 . . . −0.66 0.39 Phe 41 . . B B . . . −0.44 1.23 . . . −0.63 0.39 Gly 42 . . B B . . . −0.56 1.39 . . . −0.60 0.29 Tyr 43 . . B B . . . −0.24 1.29 . . . −0.60 0.57 Thr 44 . . B B . . . 0.31 0.90 . . . −0.45 1.14 Val 45 . . B B . . . 1.01 0.61 . . . −0.45 1.57 Gln 46 . . B B . . . 0.82 0.19 . . . −0.15 1.67 Gln 47 . . B B . . . 0.87 0.11 . . . −0.30 0.81 His 48 . . B B . . . 0.77 0.01 . . . 0.10 1.47 Asp 49 . . B B . . . 1.08 −0.20 . . F 0.95 0.84 Ile 50 . . . B T . . 1.98 −0.20 . . F 1.60 0.78 Ser 51 . . . . T T . 1.69 −0.60 . . F 2.70 1.14 Gly 52 . . . . T T . 0.88 −0.19 . . F 2.50 0.72 Asn 53 . . . . T T . 0.06 0.50 . . F 1.35 0.85 Lys 54 . . . . T T . −0.80 0.46 . . F 1.10 0.47 Trp 55 . . B B . . . −0.26 0.71 . . . −0.10 0.35 Leu 56 . . B B . . . −0.54 0.71 . . . −0.35 0.22 Val 57 . . B B . . . −0.41 0.81 . . . −0.60 0.11 Val 58 . . B B . . . −1.22 1.24 . . . −0.60 0.16 Gly 59 . . B B . . . −1.27 1.01 . . . −0.60 0.16 Ala 60 . . B . . . . −1.29 0.33 . . . −0.10 0.38 Pro 61 . . . . . . C −0.48 0.17 . * . 0.10 0.73 Leu 62 . . . . . . C 0.03 −0.07 . . F 1.34 1.19 Glu 63 A . . . . T . 0.64 −0.07 . * F 1.68 1.16 Thr 64 . . B . . T . 0.99 0.19 . * F 1.42 1.18 Asn 65 . . . . T T . 1.62 0.16 . * F 2.16 2.47 Gly 66 . . . . T T . 1.52 −0.53 . . F 3.40 2.86 Tyr 67 . . . . T . . 1.99 −0.04 . . F 2.56 2.86 Gln 68 . . . . T . . 1.99 −0.10 . . F 2.48 1.76 Lys 69 . . B . T T . 1.44 −0.50 * . F 2.60 2.97 Thr 70 . . B . . T . 1.20 −0.29 * . F 2.12 1.41 Gly 71 . . B . . T . 1.59 −0.29 * . F 2.04 1.27 Asp 72 . . B . . T . 1.17 −0.69 * . F 2.60 1.27 Val 73 . . B . . . . 0.96 −0.11 * . F 1.69 0.47 Tyr 74 . . B . . T . 0.06 −0.17 . . . 1.48 0.74 Lys 75 . . B . . T . −0.52 0.04 . . . 0.62 0.33 Cys 76 . . B . . T . −0.21 0.73 . . . 0.06 0.31 Pro 77 . . B . . T . −0.56 0.59 . . . −0.20 0.27 Val 78 . . B . . . . 0.30 0.26 . . . −0.10 0.13 Ile 79 . . B . . . . −0.12 0.66 . . . −0.40 0.40 His 80 . . B . . T . −0.48 0.66 . . . −0.20 0.14 Gly 81 . . B . . T . 0.23 0.71 . . . −0.20 0.27 Asn 82 . . . . T T . −0.37 0.07 . . . 0.50 0.77 Cys 83 . . B . . T . 0.49 0.07 . * F 0.25 0.47 Thr 84 . . B . . . . 0.57 −0.03 . * F 0.65 0.76 Lys 85 . . B . . . . 0.26 0.23 . . F 0.05 0.39 Leu 86 . . B . . . . 0.71 0.26 . . . −0.10 0.72 Asn 87 . . B . . . . −0.14 −0.31 . . . 0.50 0.97 Leu 88 . . B B . . . 0.21 −0.16 . * . 0.30 0.36 Gly 89 . . B B . . . −0.29 0.33 . * . −0.30 0.63 Arg 90 . . B B . . . −0.63 0.33 . . . −0.30 0.32 Val 91 . . B B . . . 0.18 0.31 * . . −0.30 0.53 Thr 92 . . B B . . . −0.68 0.03 * . . −0.30 0.85 Leu 93 . . B B . . . −0.17 0.24 * * . −0.30 0.32 Ser 94 . . B B . . . 0.18 0.63 . * . −0.60 0.58 Asn 95 . . B B . . . 0.18 −0.01 . * F 0.45 0.70 Val 96 . . B B . . . 1.08 −0.50 . . F 0.60 1.67 Ser 97 A . . . . . . 1.39 −1.19 * . F 1.10 2.49 Glu 98 A . . . . . . 2.20 −1.57 * . F 1.10 2.58 Arg 99 A . . . . T . 1.90 −1.57 . * F 1.30 5.60 Lys 100 A . . . . T . 2.01 −1.60 . * F 1.30 4.13 Asp 101 A . . . . T . 2.06 −1.99 . * F 1.30 4.67 Asn 102 A . . . . T . 2.01 −1.30 . * . 1.15 1.97 Met 103 A . . . . . . 1.20 −0.87 . * . 0.80 0.97 Arg 104 A . . . . . . 0.79 −0.19 . * . 0.50 0.48 Leu 105 . . B . . . . −0.07 0.20 * * . −0.10 0.40 Gly 106 . . B . . . . −0.66 0.49 * * . −0.40 0.33 Leu 107 . . B . . . . −0.97 0.37 * * . −0.10 0.17 Ser 108 . . B . . . . −0.37 0.86 * * . −0.40 0.30 Leu 109 . . B . . . . −0.69 0.57 * * . −0.06 0.49 Ala 110 . . B . . . . 0.17 0.57 . * . 0.28 0.92 Thr 111 . . B . . . . 0.51 −0.11 . . F 1.82 1.37 Asn 112 . . . . . T C 1.32 −0.50 . * F 2.56 2.78 Pro 113 . . . . T T . 1.32 −0.79 . . F 3.40 4.42 Lys 114 . . . . T T . 1.43 −0.90 . . F 3.06 4.10 Asp 115 . . . . T T . 1.21 −0.60 . . F 2.72 2.21 Asn 116 . . . . T T . 0.93 −0.31 . . F 2.08 1.18 Ser 117 . . B . . T . 0.27 −0.24 . . F 1.19 0.60 Phe 118 . . B . . T . 0.18 0.33 . . . 0.10 0.19 Leu 119 . . B . . T . −0.08 0.71 . . . −0.20 0.16 Ala 120 . . B . . . . −0.89 0.74 . . . −0.40 0.18 Cys 121 . . B . . . . −1.18 1.04 . . . −0.40 0.17 Ser 122 . . . . . T C −1.18 1.17 . . . 0.00 0.22 Pro 123 . . . . T T . −0.51 0.87 . . . 0.20 0.30 Leu 124 . . . . T T . 0.30 0.87 . . . 0.20 0.75 Trp 125 . . . . T T . 0.22 0.30 . . . 0.50 0.97 Ser 126 . . B . . . . 0.54 0.49 . . . −0.27 0.34 His 127 . . B . . T . 0.54 0.49 . . . 0.06 0.40 Glu 128 . . . . T T . 0.46 0.19 . . . 0.89 0.51 Cys 129 . . . . T T . 1.02 −0.34 . . F 1.77 0.51 Gly 130 . . . . T T . 1.07 0.03 . . F 1.30 0.59 Ser 131 . . . . T T . 1.06 0.29 . . F 1.17 0.54 Ser 132 . . . . T T . 0.78 0.77 . . F 0.89 1.44 Tyr 133 . . . . T T . 0.43 0.69 . . F 0.76 2.11 Tyr 134 . . B . . T . 0.50 0.69 . . F 0.23 1.55 Thr 135 . . B . . . . 0.18 0.91 . . F −0.10 1.15 Thr 136 . . B . . . . 0.18 1.10 . . . −0.40 0.39 Gly 137 . . B . . T . 0.59 0.73 . * . −0.20 0.34 Met 138 . . B . . T . −0.02 −0.03 . * . 0.70 0.46 Cys 139 . . B . . T . 0.22 0.13 . * . 0.10 0.23 Ser 140 . . B . . T . 0.23 0.04 . * . 0.10 0.38 Arg 141 . . B . . . . 0.54 0.00 * * F 0.05 0.52 Val 142 . . B . . . . 0.19 −0.21 * * F 0.80 1.55 Asn 143 . . B . . T . 0.90 0.00 * * F 0.61 1.00 Ser 144 . . . . . T C 0.87 −0.39 * * F 1.62 1.00 Asn 145 . . . . T T . 0.87 0.40 * * F 1.13 1.17 Phe 146 . . . . T T . 0.80 0.14 * * . 1.34 0.97 Arg 147 . . . . T . . 1.34 −0.26 * * . 2.10 1.45 Phe 148 . . . B T . . 0.49 −0.16 * * . 1.69 1.30 Ser 149 . . B B . . . 0.20 0.09 * * F 0.63 1.12 Lys 150 . . . B . . C −0.01 −0.20 * * F 1.07 0.58 Thr 151 . . . B T . . 0.10 0.23 * * F 0.61 1.03 Val 152 . . . B . . C −0.82 −0.06 * . . 0.50 0.77 Ala 153 . . B B . . C −0.12 0.24 * . . −0.10 0.32 Pro 154 A A . . . . . 0.29 0.64 * . . −0.60 0.38 Ala 155 A A . . . . . −0.42 0.16 * . . −0.15 1.01 Leu 156 A A . B . . . −0.11 0.09 * . . −0.30 0.54 Gln 157 A A . B . . . 0.43 −0.01 * . . 0.30 0.60 Arg 158 . A B B . . . 0.78 0.04 * . F −0.15 0.86 Cys 159 . A B B . . . 0.39 0.30 * * F 0.00 1.63 Gln 160 . . B B . . . 0.98 0.23 * * . −0.30 0.93 Thr 161 . . B B . . . 0.90 −0.17 * * . 0.30 0.80 Tyr 162 . . B B . . . 0.04 0.51 * * . −0.45 1.04 Met 163 . . B B . . . −0.96 0.59 . * . −0.60 0.45 Asp 164 . . B B . . . −1.14 0.87 . * . −0.60 0.22 Ile 165 . . B B . . . −1.96 1.03 . * . −0.60 0.10 Val 166 . . B B . . . −1.64 0.96 . * . −0.60 0.09 Ile 167 . . B B . . . −1.74 0.34 . * . −0.30 0.09 Val 168 . . B B . . . −1.44 0.77 . * . −0.35 0.12 Leu 169 . . B B . . . −1.44 0.47 . * . −0.10 0.22 Asp 170 . . . B T . . −0.86 0.23 * * F 1.00 0.50 Gly 171 . . . . T T . −0.89 −0.07 . . F 2.25 0.90 Ser 172 . . . . T T . −0.24 −0.03 * * F 2.50 0.77 Asn 173 . . . . . T C 0.40 0.04 * . F 1.45 0.72 Ser 174 . . . . . T C 0.92 0.47 * . F 1.05 1.12 Ile 175 . . . B . . C 0.07 0.96 * . . 0.10 0.88 Tyr 176 . . . B . . C 0.41 1.21 * . . −0.15 0.41 Pro 177 . . B B . . . −0.14 0.81 . * . −0.60 0.53 Trp 178 . . B B . . . −0.14 1.07 . * . −0.60 0.56 Val 179 . . B B . . . 0.12 0.79 . . . −0.60 0.62 Glu 180 . . B B . . . 0.31 0.53 . * . −0.60 0.54 Val 181 A . . B . . . −0.26 0.89 . . . −0.60 0.45 Gln 182 A . . B . . . −0.93 0.66 . . . −0.60 0.50 His 183 A . . B . . . −0.64 0.70 * * . −0.60 0.20 Phe 184 A . . B . . . −0.68 1.10 * . . −0.60 0.43 Leu 185 A . . B . . . −1.49 1.14 * . . −0.60 0.18 Ile 186 A . . B . . . −0.59 1.43 * . . −0.60 0.11 Asn 187 A . . B . . . −0.54 0.93 * . . −0.60 0.25 Ile 188 A . . B . . . −1.21 0.14 * * . −0.30 0.60 Leu 189 A . . B . . . −0.76 0.24 * * . −0.30 0.74 Lys 190 . . B B . . . −0.83 0.31 * . . −0.30 0.72 Lys 191 . . B B . . . −0.29 0.60 * * . −0.60 0.72 Phe 192 . . B B . . . −0.50 0.34 * . . −0.30 0.86 Tyr 193 . . B B . . . 0.04 0.09 * . . −0.30 0.67 Ile 194 . . B B . . . 0.86 0.51 * . . −0.60 0.33 Gly 195 . . . . . T C −0.08 0.91 * . . 0.00 0.66 Pro 196 . . . . T T . −0.12 0.81 . * F 0.35 0.30 Gly 197 . . . . T T . −0.28 0.46 . * F 0.35 0.73 Gln 198 . . B . . T . −0.38 0.41 . * F −0.05 0.55 Ile 199 . . B B . . . −0.34 0.41 . * . −0.60 0.35 Gln 200 . . B B . . . −0.86 0.63 . * . −0.60 0.26 Val 201 . . B B . . . −0.64 0.84 . * . −0.60 0.11 Gly 202 . . B B . . . −0.54 0.84 . * . −0.60 0.28 Val 203 . . B B . . . −0.89 0.91 . * . −0.60 0.25 Val 204 . . B B . . . 0.00 0.94 . . . −0.60 0.34 Gln 205 . . B B . . . 0.00 0.30 . . . −0.30 0.59 Tyr 206 . . B B . . . 0.00 −0.13 * . . 0.45 1.32 Gly 207 . A B B . . . −0.51 −0.13 * . F 0.60 1.32 Glu 208 A A . B . . . 0.31 −0.13 * . F 0.45 0.57 Asp 209 A A . B . . . 1.17 −0.03 * . F 0.45 0.49 Val 210 A A . B . . . 0.47 −0.79 * . . 0.60 0.86 Val 211 A A . B . . . 0.68 −0.43 * . . 0.30 0.43 His 212 A A . B . . . 0.21 0.07 * * . −0.30 0.35 Glu 213 A A . . . . . 0.21 0.76 . . . −0.60 0.39 Phe 214 A A . . . . . 0.21 0.51 . * . −0.60 0.84 His 215 A A . . . . . 0.82 −0.13 * . . 0.45 1.04 Leu 216 A A . . . . . 1.79 0.13 * . . 0.04 0.94 Asn 217 A . . . . T . 1.52 0.13 * . . 0.93 2.12 Asp 218 A . . . . T . 0.67 −0.27 * . F 2.02 2.09 Tyr 219 . . . . T T . 1.41 −0.13 * . F 2.76 1.88 Arg 220 . . . . T T . 1.44 −0.81 * . F 3.40 2.34 Ser 221 A . . . . . . 1.40 −1.21 * . F 2.46 2.34 Val 222 . A B . . . . 0.54 −0.57 * . F 1.92 1.11 Lys 223 . A B . . . . 0.54 −0.69 * . F 1.43 0.42 Asp 224 . A B . . . . 0.20 −0.69 * . F 1.09 0.54 Val 225 A A . . . . . −0.50 −0.57 * . . 0.60 0.74 Val 226 A A . . . . . −0.50 −0.71 * . . 0.60 0.37 Glu 227 A A . . . . . 0.32 −0.33 * . . 0.30 0.30 Ala 228 A A . . . . . −0.61 0.17 * . . −0.30 0.55 Ala 229 A A . . . . . −0.61 0.21 * . . −0.30 0.52 Ser 230 A A . . . . . 0.24 −0.43 * * . 0.30 0.52 His 231 A A . . . . . 1.21 −0.03 * * . 0.64 0.89 Ile 232 A A . . . . . 0.87 −0.53 * * . 1.43 1.72 Glu 233 A A . . . . . 1.11 −0.60 * * F 1.92 1.27 Gln 234 . . . . T T . 1.39 −0.56 * * F 2.91 0.92 Arg 235 . . . . T T . 1.69 −0.57 . * F 3.40 1.90 Gly 236 . . . . T T . 1.41 −1.26 * * F 3.06 1.90 Gly 237 . . . . . T C 2.41 −0.77 * * F 2.68 1.59 Thr 238 . . . . . . C 2.10 −1.17 * * F 2.30 1.59 Glu 239 . . . . . . C 1.51 −0.69 * * F 2.12 2.31 Thr 240 . . B . . . . 0.70 −0.61 * * F 1.74 2.36 Arg 241 . . B . . . . 0.70 −0.26 . * F 1.60 1.42 Thr 242 . . B . . . . 0.16 −0.31 . * F 1.29 0.81 Ala 243 A A . . . . . 0.47 0.37 . * . 0.18 0.39 Phe 244 A A . . . . . −0.23 −0.11 . * . 0.62 0.35 Gly 245 A A . . . . . −0.51 0.67 . * . −0.44 0.21 Ile 246 A A . . . . . −0.51 0.69 . * . −0.60 0.21 Glu 247 A A . . . . . −0.50 0.19 . . . −0.30 0.47 Phe 248 A A . . . . . 0.09 −0.21 . . . 0.30 0.64 Ala 249 A A . . . . . 0.20 −0.64 . . . 0.75 1.58 Arg 250 A A . . . . . −0.16 −0.83 . . F 0.75 0.92 Ser 251 A A . . . . . 0.73 −0.04 . . F 0.45 0.92 Glu 252 A A . . . . . 0.78 −0.43 . . F 0.60 1.58 Ala 253 A A . . . . . 1.13 −0.93 . . F 0.90 1.61 Phe 254 A A . . . . . 1.38 −0.50 * * F 0.60 1.19 Gln 255 A . . . . T . 1.38 −0.46 * . F 0.85 0.68 Lys 256 A . . . . T . 1.72 −0.46 * . F 1.00 1.32 Gly 257 A . . . . T . 1.38 −0.96 * . F 1.30 3.05 Gly 258 A . . . . T . 1.38 −1.31 * . F 1.30 1.74 Arg 259 A A . . . . . 2.12 −1.21 * . F 0.75 0.88 Lys 260 A A . . . . . 2.17 −1.21 * . F 0.90 1.78 Gly 261 A A . . . . . 1.27 −1.64 * . F 0.90 3.59 Ala 262 A A . . . . . 1.01 −1.43 * . F 0.90 1.36 Lys 263 A . . B . . . 0.47 −0.81 * . F 0.75 0.67 Lys 264 . . B B . . . −0.50 −0.13 * . . 0.30 0.48 Val 265 . . B B . . . −1.43 0.09 * . . −0.30 0.35 Met 266 . . B B . . . −1.40 0.27 * . . −0.30 0.12 Ile 267 . . B B . . . −0.81 0.76 * . . −0.60 0.09 Val 268 . . B B . . . −1.20 0.76 * . . −0.60 0.20 Ile 269 . . B . . T . −1.24 0.54 . . . −0.20 0.20 Thr 270 . . B . . T . −0.69 −0.07 . . F 0.85 0.49 Asp 271 . . B . . T . −0.12 −0.37 . . F 0.85 0.89 Gly 272 . . . . . T C 0.77 −0.51 . . F 1.50 1.73 Glu 273 . . . . . . C 1.32 −1.20 . . F 1.60 2.00 Ser 274 . . . . . . C 2.00 −1.30 . . F 1.90 1.60 His 275 . . . . . . C 2.31 −0.87 . . F 2.20 2.51 Asp 276 . . . . . . C 1.50 −1.30 . * F 2.50 2.42 Ser 277 . . . . . T C 1.84 −0.61 . . F 3.00 1.49 Pro 278 A . . . . T . 1.89 −1.00 . . F 2.50 1.89 Asp 279 A . . . . T . 1.33 −1.50 * . F 2.20 2.27 Leu 280 A . . . . T . 0.48 −0.86 * . F 1.90 1.25 Glu 281 A A . . . . . 0.48 −0.56 * . F 1.05 0.57 Lys 282 A A . . . . . 0.78 −0.59 * . F 0.75 0.59 Val 283 A A . . . . . 0.69 −0.19 * . . 0.45 1.24 Ile 284 A A . . . . . 0.69 −0.49 * . F 0.45 0.96 Gln 285 . A B . . . . 1.61 −0.49 * . F 0.79 0.83 Gln 286 A A . . . . . 1.61 −0.49 * . F 1.28 2.19 Ser 287 A A . . . . . 1.57 −1.13 * * F 1.92 5.22 Glu 288 . . . . . T C 1.57 −1.41 * . F 2.86 4.85 Arg 289 . . . . T T . 2.14 −1.17 * . F 3.40 2.08 Asp 290 . . . . T T . 2.26 −1.09 . . F 3.06 2.24 Asn 291 . . . . T T . 2.01 −1.47 * . F 2.72 2.53 Val 292 . . B B . . . 1.72 −0.71 . . F 1.58 2.02 Thr 293 . . B B . . . 0.87 −0.21 . . F 0.94 1.22 Arg 294 . . B B . . . 0.17 0.43 * . . −0.60 0.57 Tyr 295 . . B B . . . −0.69 0.53 * . . −0.60 0.77 Ala 296 . . B B . . . −1.50 0.53 * . . −0.60 0.40 Val 297 . . B B . . . −0.99 0.73 * . . −0.60 0.17 Ala 298 . . B B . . . −0.92 1.16 * . . −0.60 0.11 Val 299 . . B B . . . −1.28 1.16 * . . −0.60 0.16 Leu 300 . . B B . . . −1.03 1.41 * . . −0.60 0.34 Gly 301 . . B B . . . −0.33 1.17 * . . −0.60 0.55 Tyr 302 . . B B . . . 0.63 0.67 * . . −0.45 1.45 Tyr 303 . . B . . . . 0.88 0.03 * . . 0.39 3.44 Asn 304 . . B . . T . 0.84 −0.23 * . . 1.53 3.44 Arg 305 . . . . T T . 1.66 0.03 * . F 1.82 1.54 Arg 306 . . . . T T . 1.79 −0.33 * . F 2.76 1.58 Gly 307 . . . . T T . 2.03 −0.66 * . F 3.40 1.52 Ile 308 . . . . . . C 1.97 −1.06 . . F 2.66 1.34 Asn 309 . . . . . T C 1.27 −0.57 * . F 2.37 0.99 Pro 310 . . . . . T C 0.34 0.21 * . F 1.13 0.86 Glu 311 . . B . . T . 0.23 0.47 . . F 0.44 1.02 Thr 312 . . B . . T . 0.58 0.19 * . F 0.40 1.02 Phe 313 A A . . . . . 0.58 −0.21 . * . 0.45 1.14 Leu 314 A A . . . . . 0.62 0.04 * * . −0.30 0.46 Asn 315 A A . . . . . 0.59 0.04 * . . −0.30 0.64 Glu 316 A A . . . . . −0.30 0.31 * . F 0.00 1.16 Ile 317 A A . . . . . −0.58 0.21 * . . −0.30 0.98 Lys 318 A A . . . . . −0.18 0.03 * . . −0.30 0.62 Tyr 319 . A B . . . . 0.63 0.01 * . . −0.30 0.48 Ile 320 . A B . . . . 0.42 0.01 * . . −0.15 1.14 Ala 321 . A B . . . . 0.42 −0.24 * . . 0.64 0.88 Ser 322 . A B . . . . 1.31 −0.24 * . . 0.98 0.94 Asp 323 . . . . . T C 1.31 −1.00 . . F 2.52 2.23 Pro 324 A . . . . T . 1.52 −1.69 . . F 2.66 4.42 Asp 325 . . . . T T . 1.71 −1.69 . . F 3.40 4.49 Asp 326 A . . . . T . 1.60 −1.29 . . F 2.66 2.33 Lys 327 A A . . . . . 1.90 −0.50 . . F 1.62 1.30 His 328 A A . . . . . 1.04 −0.53 . * . 1.43 1.26 Phe 329 . A B . . . . 0.94 0.11 * . . 0.04 0.56 Phe 330 . A B . . . . 0.94 0.60 * . . −0.60 0.40 Asn 331 A A . . . . . 0.94 0.60 * . . −0.60 0.49 Val 332 A A . . . . . 0.31 0.10 * . . −0.30 0.99 Thr 333 A A . . . . . −0.24 −0.19 * . F 0.60 1.15 Asp 334 A A . . . . . −0.36 −0.47 * * F 0.45 0.72 Glu 335 A A . . . . . 0.39 −0.19 . * . 0.30 0.81 Ala 336 A A . . . . . 0.39 −0.83 . . . 0.75 1.12 Ala 337 A A . . . . . 0.36 −1.31 . . . 0.75 1.12 Leu 338 A A . . . . . −0.19 −0.63 * . . 0.60 0.45 Lys 339 A A . . . . . −0.19 0.01 * . . −0.30 0.33 Asp 340 A A . . . . . −0.78 −0.49 * . . 0.30 0.55 Ile 341 A A . . . . . −1.00 −0.49 * . . 0.30 0.67 Val 342 A A . . . . . −0.76 −0.49 * . . 0.30 0.28 Asp 343 A A . . . . . 0.06 −0.06 * . . 0.30 0.16 Ala 344 A A . . . . . 0.12 −0.06 * . . 0.30 0.39 Leu 345 A . . . . T . −0.77 −0.74 * . . 1.15 1.03 Gly 346 A . . . . T . −0.58 −0.70 * . . 1.00 0.43 Asp 347 A . . . . T . −0.02 0.09 * . . 0.10 0.37 Arg 348 . . B . . T . −0.83 −0.03 * . . 0.70 0.60 Ile 349 . . B . . . . −0.24 −0.03 * . . 0.50 0.50 Phe 350 . . B . . . . 0.22 −0.46 * . . 0.50 0.52 Ser 351 . . B . . . . 0.26 −0.03 * . . 0.50 0.26 Leu 352 . . B . . . . 0.26 0.46 . . . −0.10 0.54 Glu 353 . . . . . . C 0.19 0.17 . . F 1.00 1.01 Gly 354 . . . . . . C 1.08 −0.61 . . F 2.20 1.50 Thr 355 . . . . . . C 1.78 −0.60 . * F 2.50 2.93 Asn 356 . . . . . T C 1.77 −1.29 . . F 3.00 2.93 Lys 357 . . . . . T C 2.28 −0.80 . * F 2.70 4.28 Asn 358 . . . . . T C 1.58 −0.84 . . F 2.40 3.97 Glu 359 A . . . . T . 1.58 −0.54 . . F 1.90 2.14 Thr 360 A . . . . T . 1.08 −0.51 . * F 1.60 1.06 Ser 361 A . . . . T . 1.08 0.17 . * F 0.25 0.54 Phe 362 A . . . . T . 0.43 −0.23 . * . 0.70 0.54 Gly 363 A . . . . T . 0.13 0.39 . . . 0.10 0.37 Leu 364 A . . . . . . 0.13 0.29 . * . −0.10 0.37 Glu 365 A . . . . . . 0.13 0.30 . . . −0.10 0.74 Met 366 A . . . . . . 0.09 0.00 . . . 0.05 1.09 Ser 367 A . . . . T . 0.09 0.00 . . F 0.40 1.30 Gln 368 A . . . . T . 0.13 0.10 . . F 0.25 0.65 Thr 369 . . . . . T C 0.64 0.49 . . F 0.15 0.88 Gly 370 . . . . . T C 0.61 0.26 . . F 0.45 0.88 Phe 371 . . . . . . C 0.36 0.37 . . F 0.25 0.69 Ser 372 . . . B . . C −0.20 0.61 . . F −0.25 0.36 Ser 373 . . . B . . C −0.20 0.77 . . . −0.40 0.27 His 374 . . B B . . . 0.11 0.34 . . . −0.30 0.53 Val 375 . . B B . . . 0.11 −0.44 . . . 0.30 0.67 Val 376 . . B . . T . −0.04 −0.40 . . . 0.70 0.49 Glu 377 . . B . . T . −0.56 −0.14 . . . 0.70 0.27 Asp 378 A . . . . T . −1.07 0.04 . . . 0.10 0.30 Gly 379 A . . . . T . −1.38 0.09 . . . 0.10 0.33 Val 380 A A . . . . . −1.11 −0.13 . . . 0.30 0.19 Leu 381 A A . . . . . −1.11 0.37 . . . −0.30 0.11 Leu 382 . A B . . . . −1.46 1.01 . . . −0.60 0.09 Gly 383 . A B . . . . −2.04 1.01 . . . −0.60 0.11 Ala 384 . A B . . . . −1.94 0.87 . . . −0.60 0.14 Val 385 . A B . . . . −1.09 0.94 . . . −0.60 0.27 Gly 386 . . B . . . . −0.57 0.26 . . . −0.10 0.45 Ala 387 . . B . . . . 0.24 0.74 . . . −0.40 0.47 Tyr 388 . . B . . . . 0.24 0.64 . . . −0.25 1.01 Asp 389 . . . . T . . 0.24 0.43 . . . 0.15 1.01 Trp 390 A . . . . . . 0.24 0.50 . . . −0.25 1.01 Asn 391 A . . . . . . −0.22 0.64 * . . −0.40 0.48 Gly 392 A A . . . . . 0.41 0.57 * . . −0.60 0.24 Ala 393 A A . . . . . 0.66 0.57 . . . −0.60 0.45 Val 394 A A . . . . . 0.34 −0.34 . . . 0.30 0.49 Leu 395 A A . . . . . 0.33 −0.26 . . F 0.45 0.71 Lys 396 A A . . . . . −0.26 −0.30 . . F 0.45 0.94 Glu 397 A A . . . . . −0.26 −0.30 . . F 0.60 1.28 Thr 398 A A . . . . . 0.38 −0.51 . . F 0.90 1.54 Ser 399 A . . . . T . 0.38 −1.20 . . F 1.30 1.54 Ala 400 A . . . . T . 0.30 −0.56 . . F 1.15 0.66 Gly 401 A . . . . T . 0.04 0.13 . . F 0.25 0.32 Lys 402 . . B . . T . −0.77 0.07 . . F 0.25 0.37 Val 403 . . B . . . . −0.34 0.37 . . . −0.10 0.30 Ile 404 . . B . . . . −0.04 −0.13 . . . 0.50 0.60 Pro 405 . . B . . . . 0.24 −0.56 . . . 0.80 0.52 Leu 406 . . B . . . . 0.34 −0.17 . . . 0.50 0.93 Arg 407 A . . . . . . −0.51 −0.06 . . F 0.80 2.08 Glu 408 A . . . . . . 0.39 −0.06 . . F 0.80 1.11 Ser 409 A . . . . . . 1.28 −0.49 . * F 0.80 2.69 Tyr 410 A A . . . . . 0.79 −1.17 . . F 0.90 2.38 Leu 411 A A . . . . . 1.39 −0.39 . . F 0.60 1.19 Lys 412 A A . . . . . 1.28 0.04 . . F 0.00 1.37 Glu 413 A A . . . . . 1.28 −0.34 * . F 0.60 1.52 Phe 414 A A . . . . . 0.77 −1.10 * . F 0.90 3.19 Pro 415 A A . . . . . 1.06 −1.10 * . F 0.90 1.31 Glu 416 A A . . . . . 1.87 −1.10 * . F 0.90 1.52 Glu 417 A A . . . . . 1.79 −0.70 * * F 0.90 2.82 Leu 418 A A . . . . . 1.44 −0.99 * * F 0.90 2.48 Lys 419 A A . . . . . 1.56 −0.99 * * F 0.90 1.42 Asn 420 A . . . . T . 1.52 −0.49 * . . 0.70 0.83 His 421 A . . . . T . 0.71 0.27 * . . 0.25 1.57 Gly 422 A . . . . T . 0.37 0.27 . . . 0.10 0.65 Ala 423 . . B . . T . 0.93 0.70 . . . −0.20 0.40 Tyr 424 . . B B . . . 0.58 1.06 . . . −0.60 0.46 Leu 425 . . B B . . . −0.28 1.04 . . . −0.60 0.67 Gly 426 . . B B . . . −0.56 1.26 . . . −0.60 0.49 Tyr 427 . . B B . . . −0.51 1.24 . . . −0.60 0.45 Thr 428 . . B B . . . −0.78 0.87 . . . −0.60 0.74 Val 429 . . B B . . . −1.39 0.83 * . . −0.60 0.55 Thr 430 . . B B . . . −0.88 1.04 * . . −0.60 0.26 Ser 431 . . B B . . . −0.83 0.67 . . . −0.60 0.24 Val 432 . . B B . . . −0.48 0.57 . . . −0.60 0.44 Val 433 . . B B . . . −0.17 −0.07 . . F 0.79 0.60 Ser 434 . . B . . T . 0.34 −0.16 . . F 1.53 0.77 Ser 435 . . B . . T . 0.77 −0.11 . . F 2.02 1.03 Arg 436 . . B . . T . 0.21 −0.76 . . F 2.66 2.71 Gln 437 . . . . T T . 0.82 −0.76 . * F 3.40 1.50 Gly 438 . . B B . . . 0.82 −0.39 * * F 1.96 1.75 Arg 439 . . B B . . . 0.53 −0.13 * * F 1.47 0.66 Val 440 . . B B . . . 0.49 0.37 * * . 0.38 0.39 Tyr 441 . . B B . . . −0.21 0.40 * * . −0.26 0.39 Val 442 . . B B . . . −0.42 0.47 * * . −0.60 0.20 Ala 443 . . B B . . . 0.03 0.90 * * . −0.60 0.42 Gly 444 . . B B . . . −0.78 0.26 * * . −0.30 0.52 Ala 445 . . B . . . . 0.08 0.29 . * . −0.10 0.61 Pro 446 . . . . . . C 0.29 0.04 * * F 0.25 0.97 Arg 447 . . B . . . . 0.83 0.04 * * F 0.20 1.33 Phe 448 . . B . . . . 1.08 0.10 * * . 0.18 1.90 Asn 449 . . . . T . . 1.47 0.03 * * . 0.71 1.22 His 450 . . . . T T . 1.20 −0.40 * * F 1.79 1.24 Thr 451 . . . . . T C 0.52 0.24 * * F 1.12 1.06 Gly 452 . . . . T T . −0.40 0.14 * * F 1.30 0.46 Lys 453 . . B . . T . −0.40 0.43 * * F 0.47 0.28 Val 454 . . B B . . . −0.71 0.71 . . . −0.21 0.17 Ile 455 . . B B . . . −1.28 0.71 . . . −0.34 0.25 Leu 456 . . B B . . . −1.00 0.90 . . . −0.47 0.12 Phe 457 . . B B . . . −0.66 1.40 . . . −0.60 0.22 Thr 458 . . B B . . . −0.70 1.16 . . . −0.60 0.51 Met 459 . . B B . . . 0.27 0.87 . . . −0.32 1.00 His 460 . . . . . T C 0.86 0.19 . . . 1.01 2.26 Asn 461 . . . . . T C 0.86 −0.21 . . F 2.04 2.10 Asn 462 . . . . . T C 1.24 −0.01 . . F 2.32 1.75 Arg 463 . . . . T T . 0.67 −0.14 . . F 2.80 1.85 Ser 464 . . . . . . C 1.23 0.04 . . F 1.37 0.81 Leu 465 A A . . . . . 1.27 0.14 . . . 0.54 0.68 Thr 466 . A B . . . . 0.68 0.14 . . . 0.26 0.60 Ile 467 . A B . . . . 0.08 0.64 . * . −0.32 0.46 His 468 . A B . . . . 0.08 0.87 . * . −0.60 0.55 Gln 469 . A B . . . . 0.03 0.19 * * . −0.30 0.74 Ala 470 . A B . . . . 0.84 0.13 * * . 0.02 1.05 Met 471 . . B . . T . 1.16 −0.16 * . . 1.19 1.33 Arg 472 . . B . . T . 1.16 −0.26 * * F 1.51 1.33 Gly 473 . . B . . T . 0.84 0.03 * . F 0.93 0.92 Gln 474 . . B . . T . 0.54 −0.04 . . F 1.70 0.92 Gln 475 . . B . . . . 0.89 −0.27 * . F 1.33 0.63 Ile 476 . . B . . . . 0.79 0.49 * . F 0.41 1.00 Gly 477 . . B . . T . 0.33 0.84 * . F 0.29 0.50 Ser 478 . . B . . T . 0.38 0.87 * . F 0.12 0.29 Tyr 479 . . . . . T C 0.38 0.86 * . . 0.00 0.55 Phe 480 . . B . . T . −0.51 0.17 * . F 0.25 0.96 Gly 481 . . . B . . C 0.07 0.43 * . F −0.25 0.50 Ser 482 . . . B . . C 0.11 0.53 * . F −0.25 0.46 Glu 483 . . B B . . . −0.44 0.16 * . F −0.15 0.71 Ile 484 . . B B . . . −0.20 0.01 . . F −0.15 0.54 Thr 485 . . B B . . . −0.39 −0.41 . * F 0.45 0.67 Ser 486 . . B B . . . −0.04 −0.11 . * F 0.45 0.27 Val 487 . . B B . . . −0.09 −0.11 . * F 0.76 0.64 Asp 488 . . B B . . . −0.09 −0.37 . * F 1.07 0.44 Ile 489 . . B . . . . 0.46 −0.86 . * F 1.88 0.55 Asp 490 . . . . T T . −0.09 −0.81 . * F 2.79 0.73 Gly 491 . . . . T T . −0.10 −0.81 . * F 3.10 0.33 Asp 492 . . . . T T . 0.76 −0.33 * * F 2.49 0.67 Gly 493 . . B . . T . −0.10 −1.01 * * F 2.08 0.67 Val 494 . . B B . . . −0.02 −0.37 . * F 1.07 0.50 Thr 495 . . B B . . . −0.83 −0.11 . . F 0.76 0.25 Asp 496 . . B B . . . −1.34 0.57 * . F −0.45 0.21 Val 497 . . B B . . . −1.69 0.79 * . . −0.60 0.21 Leu 498 . . B B . . . −1.93 0.57 . . . −0.60 0.14 Leu 499 . . B B . . . −1.29 0.59 . . . −0.60 0.09 Val 500 . . B B . . . −1.58 1.01 . . . −0.60 0.18 Gly 501 . . B B . . . −1.82 0.99 . . . −0.60 0.21 Ala 502 . . B . . . . −1.67 1.06 . . . −0.40 0.41 Pro 503 . . B . . . . −0.86 1.16 . . . −0.40 0.48 Met 504 . . B . . . . −0.04 0.91 . . . −0.40 0.77 Tyr 505 . . B . . . . 0.47 0.49 . . . −0.25 1.33 Phe 506 . . B . . . . 0.92 0.41 * . . −0.40 0.85 Asn 507 A . . . . T . 1.51 −0.01 . * . 0.85 1.68 Glu 508 A . . . . T . 1.83 −0.63 . * F 1.30 1.86 Gly 509 A . . . . T . 2.09 −1.39 . * F 1.30 4.20 Arg 510 A . . . . T . 2.38 −1.74 . * F 1.30 2.58 Glu 511 A . . . . . . 2.22 −2.14 . * F 1.10 2.98 Arg 512 . . . B T . . 1.98 −1.50 . * F 1.30 2.24 Gly 513 . . . B T . . 1.12 −1.17 . * F 1.30 1.79 Lys 514 . . B B . . . 1.22 −0.53 . * F 0.75 0.77 Val 515 . . B B . . . 1.11 0.23 . * . −0.30 0.61 Tyr 516 . . B B . . . 0.30 0.23 . * . −0.15 1.07 Val 517 . . B B . . . 0.30 0.49 . * . −0.60 0.44 Tyr 518 . . B . . . . 0.64 0.49 * * . 0.01 1.17 Glu 519 . . B . . . . 0.60 0.24 * . . 0.57 1.29 Leu 520 . . B . . . . 1.57 −0.11 . . . 1.43 2.80 Arg 521 A . . . . T . 1.11 −0.76 . . F 2.34 3.50 Gln 522 . . B . . T . 1.11 −0.73 . . F 2.60 1.75 Asn 523 . . B . . T . 1.11 −0.09 * . F 2.04 1.57 Arg 524 . . B . . T . 1.11 −0.01 * * F 1.78 1.26 Phe 525 . . B . . . . 1.58 0.39 * . . 0.57 1.17 Val 526 . . B . . T . 1.16 0.41 * * . 0.06 0.72 Tyr 527 . . B . . T . 0.34 0.50 . * . −0.20 0.53 Asn 528 . . B . . T . 0.39 1.19 . * F 0.29 0.50 Gly 529 . . . . T T . 0.28 0.40 * * F 1.18 1.36 Thr 530 . . . . . . C 0.68 −0.24 . . F 2.02 1.45 Leu 531 . . . . . T C 1.50 −0.61 . . F 2.86 1.21 Lys 532 . . . . T T . 1.44 −0.51 . * F 3.40 1.66 Asp 533 . . B . . T . 1.20 −0.56 . * F 2.66 1.54 Ser 534 . . B . . T . 1.54 −0.29 . . F 2.02 2.93 His 535 . . B . . T . 1.86 −0.57 . . . 1.83 2.54 Ser 536 . . B . . T . 2.08 −0.17 . * . 1.19 2.44 Tyr 537 . . B . . T . 2.14 0.33 . * . 0.25 1.84 Gln 538 . . B . . T . 1.44 −0.06 . * . 0.85 2.65 Asn 539 . . B . . . . 1.40 0.23 . * . 0.05 1.71 Ala 540 . . B . . . . 1.13 0.27 . * . 0.05 1.08 Arg 541 . . B . . . . 1.13 −0.10 * * . 0.50 0.84 Phe 542 . . B . . . . 0.49 −0.11 * * F 0.65 0.70 Gly 543 . . . . T T . −0.10 0.17 * * F 0.65 0.48 Ser 544 . . . . . T C −0.40 0.17 * * F 0.45 0.25 Ser 545 . . B . . T . −0.67 0.56 . * F −0.05 0.39 Ile 546 . . B . . T . −0.67 0.41 * * . −0.20 0.29 Ala 547 . . B B . . . 0.03 −0.01 * . . 0.30 0.42 Ser 548 . . B B . . . −0.43 −0.40 . . . 0.30 0.53 Val 549 . . B B . . . −0.13 −0.10 * . . 0.64 0.62 Arg 550 . . B B . . . 0.17 −0.39 * . F 1.13 0.99 Asp 551 . . B . . . . 1.06 −0.49 * . F 1.82 1.28 Leu 552 . . B . . . . 1.34 −0.87 * . F 2.46 2.88 Asn 553 . . . . T T . 1.40 −1.13 * . F 3.40 1.97 Gln 554 . . . . T T . 2.26 −0.37 * . F 2.76 1.85 Asp 555 . . . . T T . 2.14 0.03 * . F 1.82 3.60 Ser 556 . . . . T T . 1.29 −0.66 * . F 2.38 3.74 Tyr 557 . . . B T . . 1.24 −0.41 . . F 1.34 1.60 Asn 558 . . B B . . . 0.39 −0.17 . . F 0.45 0.71 Asp 559 . . B B . . . 0.04 0.47 . . . −0.60 0.39 Val 560 . . B B . . . −0.54 0.51 * . . −0.60 0.25 Val 561 . . B B . . . −0.46 0.26 . . . −0.30 0.16 Val 562 . . B B . . . −1.02 0.29 . . . −0.30 0.14 Gly 563 . . B B . . . −1.02 0.97 . . . −0.60 0.16 Ala 564 . A B . . . . −1.02 0.33 . . . −0.30 0.38 Pro 565 A A . . . . . −0.17 −0.31 . . . 0.30 0.85 Leu 566 A A . . . . . 0.66 −0.56 . . . 0.75 1.37 Glu 567 A A . . . . . 0.92 −0.49 . . F 0.60 1.85 Asp 568 A A . . . . . 0.92 −0.49 . . . 0.45 1.21 Asn 569 A A . . . . . 0.92 −0.49 . . . 0.45 1.45 His 570 A A . . . . . 0.24 −0.67 . . . 0.60 0.85 Ala 571 A . . B . . . 0.81 0.01 . . . −0.30 0.36 Gly 572 A . . B . . . −0.08 0.77 . . . −0.60 0.35 Ala 573 A . . B . . . −0.78 1.06 . . . −0.60 0.18 Ile 574 . . B B . . . −0.81 1.34 . . . −0.60 0.15 Tyr 575 . . B B . . . −1.12 1.34 . . . −0.60 0.21 Ile 576 . . B B . . . −1.23 1.34 * * . −0.60 0.21 Phe 577 . . B B . . . −0.78 1.63 * * . −0.60 0.25 His 578 . . B B . . . −0.53 0.94 * * . −0.60 0.32 Gly 579 . . . B T . . 0.06 0.61 * * . −0.20 0.45 Phe 580 . . . . T T . −0.59 0.31 * * . 0.50 0.69 Arg 581 . . . . T T . −0.51 0.21 * * F 0.65 0.36 Gly 582 . . . . T T . 0.23 0.40 . . F 0.35 0.30 Ser 583 . . . . T T . −0.04 −0.03 . . F 1.25 0.69 Ile 584 . . . . . . C 0.09 −0.33 * * F 1.15 0.51 Leu 585 . . . . . . C 0.83 0.10 * * F 0.85 0.79 Lys 586 . . . . . . C 0.72 −0.33 . * F 1.90 1.18 Thr 587 . . . . . T C 1.18 −0.31 * * F 2.40 2.92 Pro 588 . . . . . T C 0.59 −1.00 * * F 3.00 6.94 Lys 589 . . B . . T . 1.17 −1.00 * * F 2.50 2.43 Gln 590 . . B . . T . 1.39 −0.51 * * F 2.20 2.43 Arg 591 . . B B . . . 1.04 −0.50 . * F 1.20 1.59 Ile 592 . . B B . . . 1.36 −0.54 . * F 1.20 1.06 Thr 593 . A B B . . . 0.76 −0.54 . * F 0.90 1.06 Ala 594 . A B B . . . 0.12 −0.26 . * F 0.45 0.45 Ser 595 . A B . . . . −0.19 0.24 . . F −0.15 0.65 Glu 596 . A B . . . . −0.64 0.04 . * F −0.15 0.65 Leu 597 A A . B . . . −0.57 −0.01 . . F 0.45 0.63 Ala 598 A A . B . . . −0.26 0.17 * . . −0.30 0.39 Thr 599 A A . B . . . 0.09 0.19 * . . −0.30 0.39 Gly 600 A A . B . . . −0.31 0.94 * . . −0.60 0.74 Leu 601 . . B B . . . −0.66 1.04 * . . −0.60 0.63 Gln 602 . . B B . . . −0.51 0.97 * . . −0.60 0.44 Tyr 603 . . . . T T . −0.22 1.06 * . . 0.20 0.24 Phe 604 . . B . . T . −0.80 1.01 * . . −0.20 0.38 Gly 605 . . B . . T . −0.49 1.01 * . . −0.20 0.15 Cys 606 . . B . . T . −0.02 1.11 . * . −0.20 0.13 Ser 607 . . B B . . . −0.02 0.79 . * . −0.60 0.15 Ile 608 . . B B . . . −0.59 0.40 . * . −0.60 0.27 His 609 . . B B . . . 0.11 0.66 . * . −0.60 0.41 Gly 610 . . B B . . . −0.36 0.09 . * . −0.30 0.52 Gln 611 . . B . . . . 0.31 0.39 . * . −0.10 0.61 Leu 612 . . B . . . . 0.61 0.10 . * . −0.10 0.72 Asp 613 . . B . . . . 1.50 −0.40 . * . 0.65 1.26 Leu 614 . . B . . . . 1.19 −0.83 . * . 0.95 1.21 Asn 615 A . . . . T . 0.72 −0.80 . * F 1.30 1.45 Glu 616 A . . . . T . −0.17 −0.80 . * F 1.15 0.72 Asp 617 A . . . . T . 0.64 −0.11 . * F 0.85 0.61 Gly 618 A . . . . T . −0.17 −0.80 . * F 1.15 0.63 Leu 619 A . . B . . . 0.06 −0.51 . * . 0.60 0.30 Ile 620 A . . B . . . −0.80 −0.01 . * . 0.30 0.18 Asp 621 . . B B . . . −1.14 0.63 . * . −0.60 0.14 Leu 622 . . B B . . . −1.73 0.63 * * . −0.60 0.16 Ala 623 . . B B . . . −2.20 0.44 . . . −0.60 0.24 Val 624 A . . B . . . −1.73 0.44 * . . −0.60 0.12 Gly 625 A A . . . . . −0.84 0.87 * * . −0.60 0.14 Ala 626 A A . . . . . −1.43 0.59 . . . −0.60 0.22 Leu 627 A A . . . . . −1.48 0.59 . . . −0.60 0.30 Gly 628 A A . . . . . −1.78 0.59 . . . −0.60 0.23 Asn 629 . A B B . . . −1.73 0.84 . . . −0.60 0.16 Ala 630 . A B B . . . −1.68 1.03 . . . −0.60 0.16 Val 631 . A B B . . . −1.39 1.26 * . . −0.60 0.17 Ile 632 . A B B . . . −0.47 1.21 * . . −0.60 0.14 Leu 633 . A B B . . . −0.33 0.81 * . . −0.60 0.27 Trp 634 . A B B . . . −1.19 0.74 * . . −0.60 0.57 Ser 635 . A B B . . . −1.46 0.74 * . . −0.60 0.60 Arg 636 . . B B . . . −0.60 0.70 * * F −0.45 0.54 Pro 637 . . B B . . . −0.60 0.41 * * . −0.60 0.89 Val 638 . . B B . . . 0.21 0.19 * * . −0.30 0.46 Val 639 . . B B . . . −0.09 0.20 * * . −0.30 0.38 Gln 640 . . B B . . . −0.09 0.70 * * . −0.60 0.25 Ile 641 . . B B . . . −1.01 0.66 * * . −0.60 0.45 Asn 642 . . B . . T . −0.83 0.70 . * . −0.20 0.50 Ala 643 . . B . . T . −0.68 0.56 . * . −0.20 0.39 Ser 644 . . B . . T . 0.18 0.94 . * . −0.20 0.48 Leu 645 A . . . . T . −0.03 0.26 . * . 0.10 0.52 His 646 A . . . . . . 0.56 0.29 . * . 0.18 0.80 Phe 647 A . . . . . . 0.60 0.17 . * . 0.46 0.80 Glu 648 A . . . . T . 0.30 −0.21 . * F 1.84 1.94 Pro 649 A . . . . T . 0.60 −0.21 . * F 1.97 1.00 Ser 650 . . . . T T . 0.52 −0.31 . * F 2.80 1.85 Lys 651 A . . . . T . −0.14 −0.41 . . F 1.97 0.75 Ile 652 A . . B . . . 0.52 0.37 * * F 0.69 0.42 Asn 653 A . . B . . . 0.63 0.44 * * . −0.04 0.43 Ile 654 A . . B . . . 0.84 0.06 * * . −0.02 0.42 Phe 655 . . B B . . . 0.48 0.06 * * . 0.04 1.00 His 656 . . B . . T . 0.48 −0.06 * * . 1.38 0.33 Arg 657 . . B . . T . 1.48 −0.46 * * . 1.72 0.95 Asp 658 . . . . T T . 1.18 −1.14 * . F 3.06 2.14 Cys 659 . . . . T T . 1.72 −1.54 * . F 3.40 2.11 Lys 660 . . . . T . . 2.53 −1.61 * . F 2.86 1.07 Arg 661 . . . . T T . 2.57 −1.61 * . F 3.03 1.25 Ser 662 . . . . T T . 1.87 −1.61 * . F 3.00 3.90 Gly 663 . . . . T T . 1.56 −1.69 * . F 2.97 1.97 Arg 664 . . . . T T . 1.56 −1.20 * . F 2.94 1.45 Asp 665 . . . . T T . 0.70 −0.63 * . F 3.10 0.58 Ala 666 . . B . . T . 0.00 −0.33 * . F 2.09 0.48 Thr 667 . . B . . T . −0.29 −0.26 . . . 1.63 0.25 Cys 668 . . B . . T . −0.64 0.24 * . . 0.72 0.15 Leu 669 A A . . . . . −1.57 1.03 * . . −0.29 0.13 Ala 670 A A . . . . . −2.23 1.21 . . . −0.60 0.07 Ala 671 A A . . . . . −2.34 1.30 . . . −0.60 0.07 Phe 672 A A . . . . . −2.34 1.51 . . . −0.60 0.08 Leu 673 A A . . . . . −1.89 1.31 . . . −0.60 0.11 Cys 674 . A B . . . . −1.97 1.24 . . . −0.60 0.17 Phe 675 . . B B . . . −2.08 1.43 . . . −0.60 0.14 Thr 676 . . B B . . . −2.30 1.43 . . . −0.60 0.14 Pro 677 . . B B . . . −2.19 1.43 . . . −0.60 0.22 Ile 678 . A . B T . . −1.59 1.36 . . . −0.20 0.26 Phe 679 . A B B . . . −0.96 1.00 . . . −0.60 0.28 Leu 680 . A B B . . . −0.96 1.01 . . . −0.60 0.24 Ala 681 . A . B . . C −0.64 1.37 . . . −0.40 0.30 Pro 682 . A . B . . C −0.74 1.09 . . . −0.40 0.60 His 683 . A . B T . . −0.17 0.79 . . . −0.05 1.05 Phe 684 . A . B T . . 0.22 0.59 . . . −0.05 1.51 Gln 685 . A B B . . . 0.18 0.57 . . F −0.30 1.41 Thr 686 . A B B . . . 0.42 0.79 . . F −0.45 0.77 Thr 687 . . B B . . . −0.26 0.71 * * F −0.45 0.88 Thr 688 . . B B . . . −0.11 0.61 * * F −0.45 0.35 Val 689 . . B B . . . 0.34 0.21 . * . −0.30 0.48 Gly 690 . . B B . . . 0.34 0.49 . * . −0.60 0.52 Ile 691 . . B B . . . 0.07 0.40 . * . −0.60 0.58 Arg 692 . . B B . . . 0.07 0.41 . * . −0.60 0.79 Tyr 693 . . B B . . . −0.22 0.26 . * . −0.15 1.16 Asn 694 . A B B . . . 0.63 0.44 . * . −0.45 1.63 Ala 695 . A B B . . . 0.98 −0.24 * * . 0.45 1.39 Thr 696 . A B B . . . 1.98 −0.24 . * . 0.45 1.54 Met 697 . A B B . . . 1.98 −1.00 . * F 0.90 1.87 Asp 698 . A B . . . . 1.98 −1.40 . . F 0.90 3.63 Glu 699 . A B . . . . 1.67 −1.14 . . F 1.20 3.94 Arg 700 . A B . . . . 2.04 −1.14 * * F 1.50 5.75 Arg 701 . A . . T . . 2.47 −1.33 * * F 2.20 5.33 Tyr 702 . A . . T . . 2.48 −1.33 * * F 2.50 6.02 Thr 703 . . . . . T C 2.44 −0.83 . * F 3.00 3.11 Pro 704 . . . . . T C 1.63 −0.33 . * F 2.40 2.16 Arg 705 . . B . . T . 1.52 0.36 . * . 1.15 1.14 Ala 706 . . B . . T . 1.41 −0.40 * * . 1.45 1.31 His 707 . . B . . . . 1.31 −0.89 . * . 1.59 1.47 Leu 708 . . B . . . . 1.28 −0.89 * * F 1.63 0.74 Asp 709 . . B . . T . 1.49 −0.46 . * F 1.87 0.73 Glu 710 . . . . T T . 1.49 −0.96 * * F 2.91 0.89 Gly 711 . . . . T T . 1.38 −1.46 * * F 3.40 2.12 Gly 712 . . . . T T . 1.10 −1.36 * . F 3.06 1.10 Asp 713 . . . . T . . 1.91 −0.87 * * F 2.37 0.92 Arg 714 A . . . . . . 2.02 −0.47 * * F 1.48 1.49 Phe 715 A . . . . . . 1.43 −0.90 * * F 1.44 2.95 Thr 716 . . B . . . . 0.92 −0.83 * * F 1.10 1.79 Asn 717 . A B . . . . 0.46 −0.19 * * F 0.45 0.68 Arg 718 . A B . . . . −0.36 0.50 * * F −0.45 0.64 Ala 719 . A B . . . . −0.77 0.40 * * . −0.60 0.37 Val 720 . A B . . . . −0.37 0.30 . . . −0.06 0.31 Leu 721 . A B . . . . −0.40 0.29 . . . 0.18 0.21 Leu 722 . A B . . . . −0.40 0.71 . . F 0.27 0.21 Ser 723 . . . . . T C −0.51 0.61 . . F 1.11 0.48 Ser 724 . . . . . T C −0.73 −0.03 . . F 2.40 1.01 Gly 725 . . . . . T C −0.54 −0.03 * . F 2.16 1.01 Gln 726 A . . . . T . 0.27 −0.14 * . F 1.57 0.40 Glu 727 A A . . . . . 1.19 −0.53 * * F 1.23 0.52 Leu 728 A A . . . . . 0.60 −0.91 * * F 1.14 1.03 Cys 729 A A . . . . . 0.90 −0.66 * * . 0.60 0.42 Glu 730 A A . . . . . 0.54 −0.66 * * . 0.60 0.39 Arg 731 A A . . . . . 0.51 0.13 * * . −0.30 0.41 Ile 732 A A . . . . . −0.34 −0.06 * * . 0.45 1.03 Asn 733 A A . . . . . −0.34 0.01 * * . −0.30 0.44 Phe 734 A A . . . . . 0.32 0.70 * * . −0.60 0.19 His 735 . A B . . . . 0.01 0.70 * * . −0.60 0.44 Val 736 . A B . . . . −0.69 0.50 * * . −0.60 0.40 Leu 737 . A B . . . . 0.20 0.60 . * . −0.60 0.46 Asp 738 A A . . . . . −0.04 −0.19 . . F 0.62 0.57 Thr 739 A . . . . T . −0.20 0.07 . . F 0.74 1.20 Ala 740 A . . . . T . −0.12 0.07 * . F 0.91 1.08 Asp 741 A . . . . T . 0.52 −0.61 * . . 1.83 1.30 Tyr 742 . . B . . T . 0.48 −0.19 * . . 1.70 1.39 Val 743 . . B B . . . 0.17 −0.03 . . . 1.13 1.02 Lys 744 . . B B . . . −0.22 −0.04 . . F 0.96 0.88 Pro 745 . . B B . . . 0.07 0.74 * . F −0.11 0.49 Val 746 . . B B . . . −0.79 0.37 * . . −0.13 0.88 Thr 747 . . B B . . . −0.54 0.37 * * . −0.30 0.33 Phe 748 . . B B . . . 0.07 0.37 . * . −0.30 0.37 Ser 749 . . B B . . . −0.28 0.70 . * . −0.60 0.77 Val 750 . . B B . . . −0.88 0.44 . * . −0.60 0.72 Glu 751 . . B B . . . −0.02 0.64 . * . −0.60 0.68 Tyr 752 . . B . . . . 0.29 −0.14 . * . 0.50 0.88 Ser 753 . . . . . . C 0.78 −0.53 . * . 1.49 1.99 Leu 754 . . . . T . . 1.08 −0.74 * . . 2.03 1.78 Glu 755 A . . . . . . 1.90 −0.74 * . F 2.12 1.89 Asp 756 A . . . . T . 1.56 −1.00 . . F 2.66 1.92 Pro 757 . . . . T T . 1.59 −0.96 . . F 3.40 2.31 Asp 758 . . . . T T . 1.29 −1.21 . . F 3.06 2.06 His 759 . . . . . T C 1.29 −0.60 . . F 2.52 1.22 Gly 760 . . . . . . C 1.29 0.09 . . F 0.93 0.65 Pro 761 . . B . . . . 1.29 −0.34 . . F 0.99 0.65 Met 762 . . B . . . . 1.16 −0.34 * . . 0.50 0.80 Leu 763 . . B . . . . 0.87 −0.41 * . F 0.89 0.80 Asp 764 . . . . T T . 0.69 0.07 . . F 1.13 0.54 Asp 765 . . . . T T . 0.72 0.07 . . F 1.37 0.85 Gly 766 . . . . T T . 0.62 −0.06 . . F 2.36 1.48 Trp 767 . . . . . T C 0.41 −0.26 * * F 2.40 1.28 Pro 768 . . . B . . C 1.33 0.43 * * F 0.71 0.63 Thr 769 . . B B . . . 0.48 0.43 * * F 0.42 1.25 Thr 770 . . B B . . . 0.18 0.64 * * F 0.03 0.89 Leu 771 . . B B . . . −0.33 0.11 . * . −0.06 0.77 Arg 772 . . B B . . . −0.26 0.33 * * . −0.30 0.39 Val 773 . . B B . . . −0.74 0.27 * * . −0.30 0.42 Ser 774 . . B B . . . −0.72 0.57 * * . −0.60 0.44 Val 775 . . B B . . . −0.41 0.80 * * . −0.60 0.24 Pro 776 . . B B . . . 0.06 1.20 * * . −0.60 0.52 Phe 777 . . . B T . . −0.72 0.99 * * . −0.20 0.38 Trp 778 . . . . T T . 0.13 1.17 . . . 0.20 0.28 Asn 779 . . . . . T C 0.43 0.93 . . . 0.00 0.29 Gly 780 . . . . T T . 1.29 0.50 . . . 0.20 0.57 Cys 781 . . . . T T . 1.50 −0.29 . . F 1.25 0.91 Asn 782 . A . . T . . 2.17 −1.20 . . F 1.15 0.98 Glu 783 . A . . T . . 1.79 −1.10 . . F 1.30 1.35 Asp 784 . A . . T . . 0.93 −0.96 . . F 1.30 1.35 Glu 785 . A . . T . . 1.07 −0.89 . . F 1.15 0.62 His 786 . A . . T . . 1.73 −0.86 * . . 1.00 0.56 Cys 787 A A . . . . . 0.92 −0.86 * . . 0.60 0.56 Val 788 A . . . . . . 0.07 −0.17 . . . 0.50 0.26 Pro 789 A . . . . . . −0.74 0.47 . . F −0.25 0.14 Asp 790 A A . . . . . −0.74 0.66 * . F −0.45 0.22 Leu 791 A A . . . . . −1.30 0.09 * * . −0.30 0.50 Val 792 A A . . . . . −0.52 −0.06 * * . 0.30 0.33 Leu 793 A A . . . . . 0.03 −0.49 * * . 0.30 0.38 Asp 794 . A B . . . . 0.24 −0.10 . * . 0.30 0.62 Ala 795 A A . . . . . −0.57 −0.79 . * F 0.90 1.40 Arg 796 A . . . . T . 0.03 −0.74 . * F 1.30 1.40 Ser 797 A . . . . T . 0.58 −1.00 . * F 1.30 1.29 Asp 798 A . . . . T C 0.80 −0.51 . * F 1.50 1.85 Leu 799 . . . . . T C 0.20 −0.51 * * F 1.35 0.95 Pro 800 . A . . . . C 0.79 0.10 * * F 0.05 0.70 Thr 801 A A . . . . . 0.43 −0.29 . * . 0.30 0.73 Ala 802 A A . . . . . 0.07 0.47 . . . −0.45 1.39 Met 803 A A . . . . . 0.07 0.36 * . . −0.30 0.48 Glu 804 A A . . . . . 0.99 0.33 * * . −0.30 0.58 Tyr 805 A . . B . . . 0.34 −0.16 * . . 0.45 1.12 Cys 806 A . . B . . . −0.16 −0.01 * . . 0.30 0.84 Gln 807 A . . B . . . 0.54 0.06 * . . −0.30 0.40 Arg 808 A . . B . . . 1.19 0.06 * . . −0.30 0.50 Val 809 A . . B . . . 0.98 −0.70 * . . 0.75 1.86 Leu 810 . . B B . . . 0.63 −0.84 * . F 1.20 1.66 Arg 811 . . B B . . . 1.30 −0.74 * . F 1.35 0.86 Lys 812 . . B B . . . 1.30 −0.34 * . F 1.50 2.00 Pro 813 . . . . T . . 0.52 −0.99 * . F 2.70 4.06 Ala 814 . . . . T . . 1.08 −1.10 * . F 3.00 1.11 Gln 815 . . B . . T . 1.30 −0.71 * . F 2.35 0.74 Asp 816 . . B . . T . 0.94 −0.21 * . F 1.75 0.49 Cys 817 . . B . . T . 0.59 0.11 . . . 0.70 0.75 Ser 818 . . B . . T . −0.01 0.10 . . . 0.40 0.63 Ala 819 . . B B . . . 0.28 0.39 . . . −0.30 0.31 Tyr 820 . . B B . . . −0.42 0.77 . . . −0.60 0.78 Thr 821 . . B B . . . −0.42 0.99 . . . −0.60 0.50 Leu 822 . . B B . . . −0.07 0.60 . . . −0.60 0.83 Ser 823 . . B B . . . −0.08 0.59 . * . −0.60 0.76 Phe 824 . . B B . . . −0.34 0.31 . * . −0.30 0.76 Asp 825 . . B B . . . −0.80 0.47 . * F −0.45 0.69 Thr 826 . . B B . . . −1.38 0.57 . * F −0.45 0.44 Thr 827 . . B B . . . −1.46 0.87 . * F −0.45 0.36 Val 828 . . B B . . . −1.16 0.77 . . . −0.60 0.15 Phe 829 . . B B . . . −0.76 0.77 . . . −0.60 0.18 Ile 830 . . B B . . . −1.07 0.67 . . . −0.60 0.17 Ile 831 . . B B . . . −0.64 0.67 . . . −0.60 0.33 Glu 832 A . . B . . . −0.33 0.03 . * F −0.15 0.74 Ser 833 A . . . . T . 0.63 −0.36 . * F 1.00 1.83 Thr 834 A . . . . T . 0.48 −1.04 . * F 1.30 5.10 Arg 835 A . . . . T . 0.78 −1.09 . * F 1.30 2.19 Gln 836 A . . . . T . 0.81 −0.59 . * F 1.30 1.65 Arg 837 A A . . . . . 0.81 −0.33 . * F 0.45 0.85 Val 838 . A B . . . . 0.52 −0.81 . * . 0.60 0.75 Ala 839 . A B . . . . 0.52 −0.31 . * . 0.30 0.44 Val 840 . A B . . . . −0.40 −0.23 . * . 0.30 0.32 Glu 841 A A . . . . . −0.40 0.46 . * . −0.60 0.36 Ala 842 A A . . . . . −0.51 −0.19 . * . 0.30 0.61 Thr 843 A A . . . . . 0.46 −0.29 . * . 0.45 1.33 Leu 844 A A . . . . . 0.70 −0.93 . * F 0.90 1.50 Glu 845 A A . . . . . 1.56 −0.50 . * F 0.60 1.47 Asn 846 A . . . . T . 1.56 −1.00 . * F 1.60 1.77 Arg 847 A . . . . T . 1.56 −1.09 * * F 1.90 3.45 Gly 848 A . . . . T . 1.62 −1.27 * * F 2.20 2.01 Glu 849 A . . . . T . 2.13 −0.51 * * F 2.50 1.96 Asn 850 . . . . . T C 1.82 −0.53 * . F 3.00 1.34 Ala 851 A . . . . T . 0.97 −0.04 * . F 2.20 1.95 Tyr 852 . . B . . T . 0.04 0.17 * . . 1.00 0.84 Ser 853 . . B . . T . 0.39 0.86 * . . 0.40 0.43 Thr 854 . . B B . . . −0.50 0.86 * . . −0.30 0.68 Val 855 . . B B . . . −0.80 1.04 * . . −0.60 0.31 Leu 856 . . B B . . . −0.21 0.67 * . . −0.60 0.31 Asn 857 . . B B . . . −0.27 0.69 * . . −0.60 0.37 Ile 858 . . B B . . . −0.56 0.59 * . F −0.45 0.66 Ser 859 . . B . . . . −0.24 0.44 . * F −0.25 0.81 Gln 860 . . B . . . C −0.20 0.16 . * F 0.25 0.81 Ser 861 . . . . . T C 0.61 0.44 . * F 0.15 0.96 Ala 862 . . B . . T . −0.09 0.16 . * F 0.40 1.23 Asn 863 . . B . . T . 0.21 0.56 . * . −0.20 0.62 Leu 864 A . . . . T . 0.21 0.66 . . . −0.20 0.47 Gln 865 A A . . . . . −0.60 0.66 . * . −0.60 0.62 Phe 866 A A . . . . . −1.19 0.84 . * . −0.60 0.32 Ala 867 A A . . . . . −0.60 1.13 * * . −0.60 0.27 Ser 868 A A . . . . . −0.56 0.84 * * . −0.60 0.27 Leu 869 A A . . . . . 0.26 0.44 * . . −0.60 0.62 Ile 870 A A . . . . . 0.26 −0.34 * . . 0.45 1.07 Gln 871 A A . . . . . 0.66 −0.84 * . F 1.24 1.33 Lys 872 A A . . . . . 1.24 −0.84 * . F 1.58 2.16 Glu 873 A A . . . . . 1.20 −1.53 * . F 1.92 5.14 Asp 874 . . . . T T . 1.71 −1.79 . * F 3.06 2.94 Ser 875 . . . . T T . 1.71 −1.80 . * F 3.40 1.97 Asp 876 . . . . T T . 1.71 −1.11 . * F 2.91 0.80 Gly 877 . . . . T T . 1.00 −1.11 . * F 2.57 0.83 Ser 878 A A . . . . . 0.14 −0.54 . . F 1.43 0.33 Ile 879 A A . . . . . 0.14 −0.29 . * . 0.64 0.15 Glu 880 A A . . . . . 0.44 0.11 . . . −0.30 0.24 Cys 881 . A B . . . . 0.44 −0.31 . * . 0.30 0.31 Val 882 A A . . . . . 0.90 −0.70 * . . 0.60 0.76 Asn 883 A A . . . . . 1.31 −1.39 * . F 0.75 0.86 Glu 884 A A . . . . . 1.39 −1.39 * * F 0.90 3.15 Glu 885 A A . . . . . 1.39 −1.27 * . F 0.90 3.50 Arg 886 A A . . . . . 2.10 −1.51 * . F 0.90 3.77 Arg 887 A A . . . . . 2.96 −1.91 * * F 0.90 4.35 Leu 888 A A . . . . . 2.10 −1.51 * . F 0.90 4.35 Gln 889 A A . . . . . 1.43 −0.87 * . F 0.90 1.65 Lys 890 A A . . . . . 1.43 −0.30 * . F 0.45 0.45 Gln 891 . A B . . . . 0.47 0.10 * . . −0.30 0.88 Val 892 . A B . . . . 0.06 0.06 * * . −0.30 0.38 Cys 893 . A B . . . . 0.62 0.04 * . . −0.30 0.25 Asn 894 . . B . . T . 0.41 0.80 * . . −0.20 0.23 Val 895 . . B . . T . −0.33 0.83 * . . −0.20 0.48 Ser 896 . . B . . T . −1.03 0.97 * . . −0.20 0.77 Tyr 897 . . B . . T . −0.07 1.19 * . . −0.20 0.41 Pro 898 . . B B . . . 0.01 0.79 * * . −0.45 1.09 Phe 899 A A . B . . . 0.06 0.64 * * . −0.60 0.82 Phe 900 A A . B . . . 0.32 0.26 * * . −0.15 1.05 Arg 901 A A . B . . . 0.67 0.00 * * . −0.30 0.69 Ala 902 A A . . . . . 0.06 −0.43 * * . 0.45 1.59 Lys 903 A A . . . . . −0.32 −0.57 * * F 0.90 1.36 Ala 904 A A . . . . . −0.32 −0.86 * * F 0.75 0.70 Lys 905 A A . B . . . 0.49 −0.07 * * . 0.30 0.60 Val 906 A A . B . . . −0.43 −0.57 * * . 0.60 0.59 Ala 907 A A . B . . . 0.16 0.11 . * . −0.30 0.48 Phe 908 A A . B . . . −0.59 −0.39 . * . 0.30 0.40 Arg 909 A A . B . . . 0.00 0.40 * * . −0.60 0.47 Leu 910 A A . . . . . −0.74 −0.24 * * . 0.30 0.80 Asp 911 A A . . . . . −0.19 0.04 * * . −0.30 0.80 Phe 912 A A . . . . . 0.44 −0.36 * * . 0.30 0.55 Glu 913 A A . . . . . 0.84 −0.36 * * . 0.45 1.33 Phe 914 A A . . . . . −0.16 −0.66 * * . 0.75 1.07 Ser 915 A . . . . T . −0.04 0.03 . * F 0.25 0.87 Lys 916 A . . . . T . −0.86 0.03 . * F 0.25 0.43 Ser 917 A . . . . T . −0.19 0.71 . . . −0.20 0.41 Ile 918 A . . . . T . −0.22 0.43 . . . −0.20 0.42 Phe 919 A A . . . . . −0.33 0.54 . . . −0.60 0.28 Leu 920 A A . . . . . −0.03 1.23 . . . −0.60 0.18 His 921 A A . . . . . −0.97 0.84 . * . −0.60 0.43 His 922 A A . . . . . −0.67 0.84 . * . −0.60 0.35 Leu 923 A A . . . . . −0.59 0.06 . * . −0.30 0.74 Glu 924 A A . . . . . −0.48 0.06 . * . −0.30 0.45 Ile 925 A A . . . . . −0.26 0.06 . * . −0.30 0.33 Glu 926 A A . . . . . −0.57 0.06 . * . −0.30 0.41 Leu 927 A A . . . . . −0.83 −0.20 . * . 0.30 0.23 Ala 928 A A . . . . . −0.02 0.19 . * . −0.30 0.44 Ala 929 A A . . . . . −0.32 −0.50 . * . 0.30 0.43 Gly 930 A . . . . T . 0.57 −0.11 . * F 0.85 0.69 Ser 931 . . . . . T C 0.57 −0.40 * * F 1.20 1.11 Asp 932 . . . . . T C 1.49 −0.90 . . F 1.50 1.89 Ser 933 . . . . . T C 2.08 −1.40 . . F 1.84 3.75 Asn 934 . . . . . . C 2.37 −1.83 . . F 1.98 4.67 Glu 935 A . . . . . . 2.40 −1.83 * * F 2.12 3.75 Arg 936 A . . . . . . 2.74 −1.34 . . F 2.46 4.04 Asp 937 . . . . T T . 2.74 −1.73 . * F 3.40 5.02 Ser 938 . . . . . T C 3.04 −2.13 . . F 2.86 5.02 Thr 939 A . . . . T . 3.04 −2.13 . . F 2.32 4.28 Lys 940 A . . . . T . 2.19 −1.73 * . F 1.98 4.12 Glu 941 A A . . . . . 1.49 −1.09 * . F 1.24 2.28 Asp 942 A A . . . . . 1.28 −0.97 . . F 0.90 1.60 Asn 943 A A . . . . . 0.77 −1.03 . * F 0.90 1.24 Val 944 A A . . . . . 1.19 −0.34 . * . 0.30 0.59 Ala 945 A A . . . . . 0.44 −0.34 . * . 0.30 0.69 Pro 946 A A . . . . . 0.41 0.44 . * . −0.60 0.37 Leu 947 A A . . . . . −0.40 0.54 . * . −0.60 0.68 Arg 948 A A . . . . . −0.36 0.59 . * . −0.60 0.56 Phe 949 A A . . . . . 0.26 0.09 . * . −0.30 0.72 His 950 A A . . . . . 0.84 0.41 . * . −0.45 1.37 Leu 951 A A . . . . . 0.47 −0.27 * * . 0.45 1.21 Lys 952 A A . . . . . 1.28 0.23 * * . −0.15 1.41 Tyr 953 A A . . . . . 0.31 −0.56 * * . 0.75 1.73 Glu 954 A A . . . . . 0.20 −0.41 . * . 0.45 1.56 Ala 955 A . . B . . . −0.47 −0.41 . * . 0.30 0.64 Asp 956 A . . B . . . 0.03 0.37 . * . −0.30 0.35 Val 957 A . . B . . . 0.10 0.10 . * . −0.30 0.30 Leu 958 A . . B . . . 0.04 0.10 . . . −0.30 0.57 Phe 959 A . . B . . . −0.26 −0.01 . . . 0.30 0.46 Thr 960 A . . B . . . 0.03 0.37 . . F 0.06 0.83 Arg 961 A . . B . . . −0.78 0.11 . . F 0.42 1.35 Ser 962 . . . . T T . −0.22 0.11 . . F 1.43 1.29 Ser 963 . . . . . T C 0.56 −0.29 . . F 2.04 1.19 Ser 964 . . . . . T C 1.01 −0.27 . . F 2.10 0.83 Leu 965 . . . . . T C 1.32 0.49 . . F 0.99 0.97 Ser 966 . . . . . . C 0.36 0.10 . * . 0.88 1.25 His 967 . . B . . . . 0.70 0.36 . * . 0.32 0.69 Tyr 968 . . B . . . . 0.19 −0.03 . * . 0.86 1.68 Glu 969 . . B . . . . 0.49 −0.03 . * . 0.65 1.04 Val 970 A . . . . . . 1.00 −0.01 . * . 0.65 1.22 Lys 971 A . . . . . . 1.00 −0.13 . * F 0.80 1.05 Leu 972 A . . . . . . 0.22 −0.50 . * F 0.65 0.81 Asn 973 . . . . . T C 0.47 0.19 * * F 0.45 0.90 Ser 974 A . . . . T . 0.58 −0.46 * * F 0.85 0.78 Ser 975 A . . . . T . 1.19 −0.46 * * F 1.00 1.85 Leu 976 . . B . . T . 1.14 −0.39 * * F 1.31 1.80 Glu 977 . . B . . . . 1.61 −0.79 * . F 1.72 2.25 Arg 978 . . B . . T . 0.72 −0.74 * . F 2.23 1.66 Tyr 979 . . B . . T . 0.68 −0.44 . . F 2.24 1.41 Asp 980 . . . . T T . 0.77 −0.70 . . F 3.10 0.81 Gly 981 . . . . T T . 1.37 −0.27 * . F 2.49 0.64 Ile 982 . . . . T . . 0.67 0.16 * . F 1.38 0.63 Gly 983 . . . . . . C 0.26 0.19 . * F 0.87 0.33 Pro 984 . . . . . T C −0.17 0.57 * . F 0.46 0.44 Pro 985 . . . . T T . −1.06 0.71 * . F 0.35 0.34 Phe 986 . . . . T T . −1.41 0.71 * * . 0.20 0.24 Ser 987 . . B . . T . −0.41 1.07 * * . −0.20 0.13 Cys 988 . . B B . . . −0.96 0.64 * * . −0.60 0.17 Ile 989 . . B B . . . −0.74 0.90 * * . −0.60 0.14 Phe 990 . . B B . . . −0.53 0.51 * * . −0.60 0.18 Arg 991 . . B B . . . −0.64 0.53 * * . −0.60 0.53 Ile 992 . . B B . . . −0.69 0.64 * * . −0.60 0.63 Gln 993 . . B B . . . −0.83 0.39 * * . −0.30 0.72 Asn 994 . . . B T . . −0.64 0.29 * * . 0.10 0.30 Leu 995 . . . B T . . −0.16 1.07 . * . −0.20 0.37 Gly 996 . . . B T . . −1.16 0.81 * * . −0.20 0.33 Leu 997 . . . . . . C −0.30 1.10 . . . −0.20 0.14 Phe 998 . . B . . . . −0.64 1.20 . . . −0.40 0.24 Pro 999 . . B . . . . −1.53 0.94 . . . −0.40 0.24 Ile 1000 A . . B . . . −1.32 1.20 . . . −0.60 0.20 His 1001 A . . B . . . −1.58 1.13 . . . −0.60 0.23 Gly 1002 A . . B . . . −0.72 0.96 * . . −0.60 0.15 Ile 1003 A . . B . . . −0.91 0.53 . * . −0.60 0.42 Met 1004 A . . B . . . −1.01 0.53 . * . −0.60 0.22 Met 1005 . . B B . . . −1.01 0.51 . * . −0.60 0.32 Lys 1006 . . B B . . . −1.19 0.77 . * . −0.60 0.32 Ile 1007 . . B B . . . −1.73 0.51 . * . −0.60 0.50 Thr 1008 . . B B . . . −1.43 0.59 . * . −0.60 0.35 Ile 1009 . . B B . . . −1.14 0.47 * * . −0.60 0.18 Pro 1010 . . B B . . . −0.43 0.96 * * . −0.60 0.37 Ile 1011 . . B B . . . −0.78 0.27 * * . 0.04 0.50 Ala 1012 . . B B . . . −0.23 0.17 * * . 0.38 0.95 Thr 1013 . . B . . T . 0.08 −0.09 * * F 1.87 0.61 Arg 1014 . . . . T T . 1.08 −0.11 * * F 2.76 1.40 Ser 1015 . . . . T T . 0.48 −0.80 * * F 3.40 2.71 Gly 1016 . . . . T T . 0.56 −0.61 * * F 3.06 1.55 Asn 1017 . A . . T . . 1.19 −0.41 * . F 1.87 0.65 Arg 1018 . A B . . . . 0.69 −0.41 * * F 1.13 0.97 Leu 1019 . A B . . . . 0.69 −0.11 * * F 0.79 0.81 Leu 1020 . A B . . . . 0.99 −0.54 . . F 0.75 0.99 Lys 1021 . A B . . . . 0.63 −0.94 * . F 0.75 0.84 Leu 1022 . A B . . . . −0.18 −0.16 * . F 0.45 0.88 Arg 1023 . A B . . . . −0.60 −0.16 * . F 0.45 0.88 Asp 1024 . A B . . . . 0.21 −0.36 * * F 0.45 0.64 Phe 1025 . A B . . . . 1.02 −0.36 * * . 0.45 1.29 Leu 1026 A A . . . . . 0.12 −1.04 * * F 0.90 1.14 Thr 1027 A A . . . . . 0.34 −0.40 * . F 0.45 0.51 Asp 1028 A A . . . . . 0.23 0.10 . . F −0.15 0.59 Glu 1029 A A . . . . . −0.08 −0.29 * . . 0.45 1.15 Val 1030 A A . . . . . 0.32 −0.49 . . . 0.45 1.15 Ala 1031 A A . . . . . 0.47 −0.59 * . . 0.60 0.93 Asn 1032 . . . . T T . 0.78 −0.01 * . F 1.25 0.29 Thr 1033 . . . . T T . −0.11 0.39 * . F 0.65 0.62 Ser 1034 . . B . . T . −0.40 0.43 * . F −0.05 0.43 Cys 1035 . . B . . T . 0.11 0.84 * * . −0.20 0.28 Asn 1036 . . . . T . . 0.70 0.87 . * . 0.16 0.19 Ile 1037 . . . . T . . 0.40 0.79 . * . 0.32 0.23 Trp 1038 . . . . T T . 0.40 0.79 . . . 0.68 0.58 Gly 1039 . . . . . T C 0.70 0.70 . . F 0.79 0.52 Asn 1040 . . . . T T . 1.12 0.30 . * F 1.60 1.28 Ser 1041 . . . . . T C 1.23 0.37 . * F 1.24 1.91 Thr 1042 . . . . . . C 1.91 −0.54 . * F 1.78 3.79 Glu 1043 . . . . T . . 1.89 −0.54 . * F 1.82 3.64 Tyr 1044 . . . . T . . 2.02 −0.46 . . F 1.66 3.92 Arg 1045 . . . . . T C 1.17 −0.41 . . F 1.80 4.20 Pro 1046 . . . . . T C 1.47 −0.26 . . F 2.10 1.80 Thr 1047 . . . . . T C 1.78 −0.26 . . F 2.40 1.99 Pro 1048 . . . . . T C 1.78 −1.01 . * F 3.00 1.76 Val 1049 A A . . . . . 1.21 −1.01 . * F 2.10 1.90 Glu 1050 A A . . . . . 1.21 −0.76 * . F 1.80 1.09 Glu 1051 A A . . . . . 1.53 −1.24 * . F 1.50 1.38 Asp 1052 A A . . . . . 1.26 −1.67 * . F 1.20 3.63 Leu 1053 A A . . . . . 1.26 −1.81 * . F 0.90 2.12 Arg 1054 A A . . . . . 2.11 −1.39 * * F 0.90 1.89 Arg 1055 A A . . . . . 1.30 −0.99 * * F 0.90 1.96 Ala 1056 A A . . . . . 1.30 −0.30 * . F 0.60 1.96 Pro 1057 A A . . . . . 1.27 −0.59 * * F 0.90 1.61 Gln 1058 A A . . . . . 1.78 −0.09 * . F 0.60 1.12 Leu 1059 . A B . . . . 1.67 0.30 * . . 0.13 1.48 Asn 1060 . A . . . . C 1.26 0.20 . . . 0.61 1.54 His 1061 . . . . . T C 1.84 0.16 . . F 1.44 1.19 Ser 1062 . . . . . T C 1.20 −0.24 . . F 2.32 2.42 Asn 1063 . . . . T T . 0.34 −0.29 * . F 2.80 1.12 Ser 1064 . . . . T T . 0.86 −0.04 * . F 2.37 0.61 Asp 1065 . . B B . . . −0.03 −0.16 . . F 1.29 0.61 Val 1066 . . B B . . . 0.00 0.14 . . F 0.41 0.27 Val 1067 . . B B . . . −0.37 0.14 . . . −0.02 0.32 Ser 1068 . . B . . T . −0.37 0.33 . * . 0.10 0.10 Ile 1069 . . B . . T . −0.96 0.73 * * . −0.20 0.22 Asn 1070 . . B . . T . −0.84 0.77 . * . −0.20 0.21 Cys 1071 . . B . . T . −0.80 0.13 * * . 0.10 0.31 Asn 1072 . . B B . . . −0.80 0.43 * * . −0.60 0.36 Ile 1073 . . B B . . . −0.71 0.39 * * . −0.30 0.17 Arg 1074 . . B B . . . 0.18 0.41 * * . −0.60 0.48 Leu 1075 . . B B . . . 0.18 0.24 * * . −0.30 0.48 Val 1076 . . B . . T . 0.84 0.24 . * F 0.40 1.18 Pro 1077 . . . . . T C −0.04 −0.44 * * F 1.20 1.05 Asn 1078 . . . . T T . 0.84 0.24 * * F 0.65 0.89 Gln 1079 A . . . . T . 0.03 −0.04 * * F 1.00 1.93 Glu 1080 . A B . . . . 0.81 0.10 . * F 0.00 1.08 Ile 1081 . A B . . . . 0.86 0.17 . * F −0.15 0.91 Asn 1082 . A B . . . . 0.26 0.46 . * . −0.60 0.43 Phe 1083 . A B . . . . −0.09 0.74 . * . −0.60 0.21 His 1084 . A B . . . . −0.09 1.17 . * . −0.60 0.29 Leu 1085 . A B . . . . −0.90 0.89 . * . −0.60 0.29 Leu 1086 . A . . . . C −0.30 1.17 . * . −0.40 0.28 Gly 1087 . A . . T . . −1.11 1.30 * * . −0.20 0.22 Asn 1088 A A . . . . . −0.30 1.49 * * . −0.60 0.22 Leu 1089 A A . . . . . −0.57 0.80 * * . −0.60 0.51 Trp 1090 A A . . . . . −0.57 0.50 * * . −0.60 0.69 Leu 1091 A A . . . . . 0.29 0.76 * . . −0.60 0.35 Arg 1092 A A . . . . . 0.04 0.36 * * . −0.30 0.86 Ser 1093 A A . . . . . −0.77 0.17 * . . −0.30 0.83 Leu 1094 A A . . . . . 0.09 −0.06 * . F 0.45 0.83 Lys 1095 A A . . . . . 0.13 −0.74 * . F 0.75 0.84 Ala 1096 A A . . . . . 0.99 0.01 * * . −0.30 0.99 Leu 1097 A A . . . . . 0.58 −0.37 * . . 0.45 2.39 Lys 1098 A A . . . . . 0.28 −0.67 . . F 0.90 1.60 Tyr 1099 A A . . . . . 1.13 −0.06 * . F 0.60 1.57 Lys 1100 A A . . . . . 0.20 −0.56 * . F 0.90 3.81 Ser 1101 A A . . . . . 0.19 −0.56 . * F 0.90 1.34 Met 1102 A A . . . . . 0.14 0.06 . * . −0.30 0.84 Lys 1103 A A . . . . . 0.10 −0.06 . * . 0.30 0.31 Ile 1104 . A B . . . . −0.24 0.34 * * . −0.30 0.38 Met 1105 A A . . . . . −0.88 0.46 * * . −0.60 0.38 Val 1106 A A . . . . . −1.39 0.34 * * . −0.30 0.19 Asn 1107 A A . . . . . −0.79 1.03 . * . −0.60 0.23 Ala 1108 A A . . . . . −0.72 0.74 . * . −0.60 0.40 Ala 1109 A A . . . . . 0.17 0.13 . * . −0.15 1.05 Leu 1110 A A . . . . . 0.07 −0.11 * . . 0.45 1.13 Gln 1111 A A . . . . . 0.89 0.27 * . . −0.30 0.97 Arg 1112 A A . . . . . 0.59 0.27 * . . −0.15 1.31 Gln 1113 . A B . . . . 0.97 0.16 * . . −0.15 2.13 Phe 1114 . A . . T . . 0.86 −0.10 * . . 0.85 1.90 His 1115 . A . . . . C 0.78 0.29 * . . −0.10 0.84 Ser 1116 . A . . . . C 0.08 0.97 . * . −0.40 0.34 Pro 1117 . . . B . . C 0.08 1.36 . * . −0.40 0.34 Phe 1118 . A . B . . C 0.08 0.57 . * . −0.40 0.49 Ile 1119 . A B B . . . 0.78 0.07 . * . −0.30 0.63 Phe 1120 . A B B . . . 0.81 −0.31 . . . 0.30 0.71 Arg 1121 A A . B . . . 0.90 −0.74 . . F 1.24 1.37 Glu 1122 . A . . T . . 0.81 −1.10 * * F 1.98 3.01 Glu 1123 . A . . . . C 1.62 −1.40 * * F 2.12 4.66 Asp 1124 . . . . . T C 2.51 −2.19 * . F 2.86 4.66 Pro 1125 . . . . T T . 2.32 −1.79 * * F 3.40 4.66 Ser 1126 . . . . T T . 1.36 −1.10 * . F 3.06 1.89 Arg 1127 A . . . . T . 0.66 −0.46 . . F 1.87 0.84 Gln 1128 A . . B . . . 0.66 0.33 * . F 0.53 0.47 Ile 1129 A . . B . . . −0.23 −0.10 * . . 0.64 0.61 Val 1130 A . . B . . . −0.32 0.20 * . . −0.30 0.22 Phe 1131 . . B B . . . 0.02 0.59 * . . −0.60 0.17 Glu 1132 . . B B . . . −0.09 0.19 * . . −0.30 0.48 Ile 1133 . . B B . . . −0.09 −0.10 * * F 0.60 1.12 Ser 1134 . A . . . . C 0.80 −0.74 . * F 1.10 2.24 Lys 1135 . A . . T . . 1.37 −1.53 . . F 1.30 2.16 Gln 1136 . A . . T . . 2.07 −0.61 . * F 1.30 3.24 Glu 1137 A A . . . . . 1.21 −0.90 . * F 0.90 4.18 Asp 1138 . A . B T . . 1.89 −0.64 . * F 1.30 1.55 Trp 1139 . A . B T . . 1.30 −0.21 . * . 0.85 1.39 Gln 1140 . A B B . . . 0.97 0.07 . * . −0.30 0.56 Val 1141 . A B B . . . 0.08 0.99 . * . −0.60 0.35 Pro 1142 . A B B . . . −0.81 1.67 . * . −0.60 0.24 Ile 1143 . . B B . . . −1.67 1.44 . * . −0.60 0.10 Trp 1144 . . B B . . . −1.72 1.69 . * . −0.60 0.10 Ile 1145 . . B B . . . −2.02 1.47 . . . −0.60 0.06 Ile 1146 . . B B . . . −1.48 1.43 . . . −0.60 0.12 Val 1147 . . B B . . . −2.08 1.23 . . . −0.60 0.16 Gly 1148 . . B B . . . −1.53 1.00 . . F −0.45 0.19 Ser 1149 . . . B . . C −1.59 0.74 . . F −0.25 0.27 Thr 1150 . . . B . . C −1.51 0.49 . . F −0.25 0.35 Leu 1151 . . . B . . C −1.43 0.53 . . F −0.25 0.30 Gly 1152 . . . B T . . −1.39 0.79 . . F −0.05 0.18 Gly 1153 . A B B . . . −1.86 1.09 . . . −0.60 0.10 Leu 1154 . A B B . . . −2.14 1.29 . . . −0.60 0.10 Leu 1155 . A B B . . . −2.64 1.10 . . . −0.60 0.11 Leu 1156 A A . B . . . −2.64 1.36 . . . −0.60 0.09 Leu 1157 A A . B . . . −3.16 1.61 . . . −0.60 0.09 Ala 1158 A A . B . . . −3.62 1.57 . . . −0.60 0.08 Leu 1159 A A . B . . . −3.40 1.57 . . . −0.60 0.08 Leu 1160 A A . B . . . −3.40 1.39 . . . −0.60 0.10 Val 1161 A A . B . . . −2.88 1.39 . . . −0.60 0.08 Leu 1162 A A . B . . . −2.02 1.80 * . . −0.60 0.10 Ala 1163 A A . B . . . −2.24 1.11 . . . −0.60 0.25 Leu 1164 A A . B . . . −1.78 1.11 . * . −0.60 0.27 Trp 1165 A A . B . . . −1.67 0.90 . . . −0.60 0.33 Lys 1166 A A . B . . . −1.51 1.00 . . . −0.60 0.28 Leu 1167 A A . B . . . −0.59 1.29 . . . −0.60 0.29 Gly 1168 A . . B . . . −0.30 0.60 . . . −0.60 0.55 Phe 1169 . . B B . . . −0.08 0.07 * . . −0.30 0.37 Phe 1170 . . B B . . . 0.32 0.57 * . . −0.60 0.45 Arg 1171 . . B B . . . 0.39 −0.11 * . . 0.30 0.89 Ser 1172 . . . B . . C 1.31 −0.54 * . F 1.10 2.02 Ala 1173 . . . . . . C 1.77 −1.33 * . F 1.30 4.57 Arg 1174 . . . . . . C 2.47 −2.11 * . F 1.30 4.57 Arg 1175 . . . . T . . 2.96 −2.11 * . F 1.84 5.90 Arg 1176 . . . . T . . 2.50 −2.07 * . F 2.18 9.03 Arg 1177 . . . . T . . 1.99 −2.14 . . F 2.52 4.56 Glu 1178 . . . . . T C 2.58 −1.46 . . F 2.86 1.92 Pro 1179 . . . . T T . 2.26 −1.46 . . F 3.40 1.64 Gly 1180 . . . . T T . 1.83 −1.03 . . F 3.06 1.29 Leu 1181 . . . . . T C 1.51 −0.54 . * F 2.69 1.08 Asp 1182 . . . . . T C 1.44 −0.11 . * F 2.22 1.08 Pro 1183 . . . . . T C 0.59 −0.54 * * F 2.35 2.18 Thr 1184 . . . . . T C −0.01 −0.33 * . F 1.88 1.96 Pro 1185 . . B . . T . 0.33 −0.33 * . F 1.70 0.97 Lys 1186 . A B . . . . 0.76 −0.33 * * F 1.28 1.08 Val 1187 . A B . . . . 0.37 −0.33 * . F 0.96 0.96 Leu 1188 A A . . . . . 0.19 −0.39 * . . 0.64 0.79 Glu 1189 A A . . . . . 0.11 −0.39 * . . 0.47 0.51

INCORPORATION BY REFERENCE

The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background of the Invention, Detailed Description, and Examples is hereby incorporated herein by reference. In addition, the sequence listing submitted herewith is incorporated herein by reference in its entirety. The specification and sequence listing of each of the following U.S. and PCT applications are herein incorporated by reference in their entirety: U.S. Provisional Appln. No. 60/243,792 filed on Oct. 30, 2000, U.S. Provisional Appln. No. 60/198,407 filed on Apr. 19, 2000, U.S. Provisional Appln. No. 60/105,971 filed on Oct. 28, 1998, U.S. application Ser. No. 09/836,353 filed on Apr. 18, 2001, PCT Appln. No. PCT/US99/25031 filed on Oct. 27, 1999, U.S. Provisional Appln. No. 60/304,417 filed on Jul. 12, 2001, U.S. Provisional Appln. No. 60/270,625 filed on Feb. 23, 2001, U.S. Provisional Appln. No. 60/277,340 filed on 21 Mar. 2001, U.S. Provisional Appln. No. 60/306,171 filed on 19 Jul. 2001, U.S. Provisional Appln. No. 60/278,650 filed on 27 Mar. 2001, U.S. Provisional Appln. No. 60/331,287 filed on 13 Nov. 2001, U.S. application Ser. No. 09/950,082 filed on 12 Sep. 2001, U.S. application Ser. No. 09/950,083 filed on 12 Sep. 2001, U.S. Provisional Appln. No. 60/040,162 filed on 7 Mar. 1997, U.S. Provisional Appln. No. 60/043,576 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,601 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,845 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,580 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,599 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,664 filed on 22 Aug. 1997, U.S. Provisional Appln. No. 60/043,314 filed on 11 Apr. 1997, U.S. Provisional Appln. No. 60/047,632 filed on 23 May 1997, U.S. Provisional Appln. No. 60/056,892 filed on 22 Aug. 1997, U.S. Provisional Appln. 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What is claimed is:
 1. A nucleic acid molecule comprising a polynucleotide sequence selected from the group consisting of: (a) a polynucleotide fragment of SEQ ID NO:X as referenced in Table 1A; (b) a polynucleotide encoding a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (c) a polynucleotide encoding a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (d) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (e) a polynucleotide encoding a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity; (f) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B.1; (g) a polynucleotide encoding a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; (h) a polynucleotide encoding a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.1; and (i) a polynucleotide which is at least 95% identical to a polynucleotide as defined in any one of (a)-(h).
 2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a heterologous polynucleotide sequence.
 3. A recombinant vector comprising the nucleic acid molecule of claim
 1. 4. A recombinant vector comprising the nucleic acid molecule of claim
 2. 5. A recombinant host cell comprising the recombinant vector of claim
 3. 6. A recombinant host cell comprising the recombinant vector of claim
 4. 7. Use of the nucleic acid molecule of claim 1 for the preparation of a diagnostic or pharmaceutical composition for diagnosing or treating a medical condition.
 8. A polypeptide comprising an amino acid sequence selected from the group consisting of: (a) a full length polypeptide of SEQ ID NO:Y or a full length polypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (b) a predicted secreted form of SEQ ID NO:Y or a secreted form of the polypeptide encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (c) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A; (d) a polypeptide fragment of SEQ ID NO:Y or a polypeptide fragment encoded by the cDNA Clone ID in ATCC Deposit No:Z corresponding to SEQ ID NO:Y as referenced in Table 1A, wherein said fragment has biological activity; (e) a polypeptide domain of SEQ ID NO:Y as referenced in Table 1B.1; (f) a polypeptide domain of SEQ ID NO:Y as referenced in Table 2; (g) a predicted epitope of SEQ ID NO:Y as referenced in Table 1B.1; and (h) a polypeptide which is at least 95% identical to a polypeptide as defined in any one of (a)-(g).
 9. The polypeptide of claim 8, wherein said polypeptide comprises a heterologous amino acid sequence.
 10. Use of the polypeptide of claim 8 for identifying a binding partner comprising: (a) contacting the polypeptide of claim 8 with a binding partner; and (b) determining whether the binding partner increases or decreases activity of the polypeptide.
 11. Use of the polypeptide of claim 8 for the preparation of a diagnostic or pharmaceutical composition for diagnosing or treating a medical condition.
 12. An antibody or fragment thereof that specifically binds the polypeptide of claim
 8. 13. Use of the antibody or fragment thereof of claim 11 for the preparation of a diagnostic or pharmaceutical composition for diagnosing or treating a medical condition.
 14. Use of an agonist or antagonist of the polypeptide of claim 8 for the preparation of a pharmaceutical composition for treating a medical condition. 